B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3

available at www.sciencedirect.com

www.elsevier.com/locate/brainresrev

Review

Neuroinflammation and brain infections: Historical context

and current perspectives

Marina Bentivoglio⁎, Raffaella Mariotti, Giuseppe Bertini

Department of Neurological, Neuropsychological, Morphological and Motor Sciences, University of Verona, Verona, Italy
National Institute of Neuroscience, Italy

A R T I C LE I N FO AB S T R A C T

Article history: An overview of current concepts on neuroinflammation and on the dialogue between neurons
Accepted 22 September 2010 and non-neuronal cells in three important infections of the central nervous systems (rabies,
Available online 29 September 2010 cerebral malaria, and human African trypanosomiasis or sleeping sickness) is here presented.
Large numbers of cases affected by these diseases are currently reported. In the context of an
Keywords: issue dedicated to Camillo Golgi, historical notes on seminal discoveries on these diseases are
Rabies also presented. Neuroinflammation is currently closely associated with pathogenetic
Cerebral malaria mechanisms of chronic neurodegenerative diseases. Neuroinflammatory signaling in brain
Human African trypanosomiasis infections is instead relatively neglected in the neuroscience community, despite the fact
Astrocytes that the above infections provide paradigmatic examples of alterations of the intercellular
Microglia crosstalk between neurons and non-neuronal cells. In rabies, strategies of immune evasion of
Lymphocytes the host lead to silencing neuroinflammatory signaling. In the intravascular pathology which
Neurodegeneration characterizes cerebral malaria, leukocytes and Plasmodium do not enter the brain parenchyma.
Non-cell autonomous In sleeping sickness, leukocytes and African trypanosomes invade the brain parenchyma at an
advanced stage of infection. Both the latter pathologies leave open many questions on the
targeting of neuronal functions and on the pathogenetic role of non-neuronal cells, and in
particular astrocytes and microglia, in these diseases. All three infections are hallmarked by
very severe clinical pictures and relative sparing of neuronal structure. Multidisciplinary
approaches and a concerted action of the neuroscience community are needed to shed light on
intercellular crosstalk in these dreadful brain diseases. Such effort could also lead to new
knowledge on non-neuronal mechanisms which determine neuronal death or survival.
© 2010 Published by Elsevier B.V.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
1.1. Neuroinflammation: an old concept with novel implications . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
1.1.1. Immune specialization versus immune privilege within the CNS . . . . . . . . . . . . . . . . . . . . . 153
1.1.2. Activation of astrocytes and microglia in the immune response . . . . . . . . . . . . . . . . . . . . . 154
1.1.3. The growing interest in the neuroinflammatory component of neurodegenerative diseases . . . . . . 156
1.1.4. The concept of “non-cell autonomous” neurodegenerative disease . . . . . . . . . . . . . . . . . . . . 156

⁎ Corresponding author. Dip. Scienze Neurologiche, Facoltà di Medicina, Strada Le Grazie 8, 37134 Verona, Italy. Fax: + 39 045 8027163.
E-mail address: marina.bentivoglio@univr.it (M. Bentivoglio).

0165-0173/$ – see front matter © 2010 Published by Elsevier B.V.

doi:10.1016/j.brainresrev.2010.09.008
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3 153

2. The fight of rabies virus against neuroinflammation: Evading the immune response . . . . . . . . . . . . . . . . . 157
2.1. The discovery of Negri bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
2.2. The discovery of centrifugal spread of rabies virus to the salivary glands . . . . . . . . . . . . . . . . . . . . 159
2.3. Rabies today . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
2.3.1. Rabies virus transmission and clinical features of the infection . . . . . . . . . . . . . . . . . . . . . 159
2.3.2. Neuropathology of rabies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160
2.3.3. Negri bodies today and evasion of the immune response in rabies . . . . . . . . . . . . . . . . . . . 161
2.4. Rabies virus as tract tracing tool . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
3. Malaria and the brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
3.1. Malaria today . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
3.2. Camillo Golgi and malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
3.3. Cerebral malaria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
3.3.1. The puzzle of the pathology and pathogenesis of cerebral malaria . . . . . . . . . . . . . . . . . . . 165
4. Human African trypanosomiasis: sleep and wake in flame. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
4.1. HAT today . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
4.2. Historical note . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
4.3. Neuropathology of African trypanosomiasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
4.4. Neuroinflammatory signaling and African trypanosome infection . . . . . . . . . . . . . . . . . . . . . . . . 167
5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

1. Introduction neuroscience (see in this issue Verkhratsky et al., 2010),

The present overview aims at summarizing data on the cross-
talk between neurons and non-neuronal cells that characterize
three severe infections of the central nervous system (CNS):
rabies, cerebral malaria, and human African trypanosomiasis
(HAT). Cerebral malaria is one the most common encephalo-
pathies worldwide and is widely regarded as the most important
parasitic CNS disease. Rabies and HAT are neglected diseases in
spite of the large numbers of currently reported cases.
These three diseases provide different and paradigmatic
examples of marked disturbances of neuronal function caused
by the induction and/or downregulation of neuroinflamma-
tory signaling. Remarkably, all three diseases share the
absence of overt degenerative features of neuronal cell
populations. Thus, although much attention has recently
been devoted to the role of chronic neuroinflammation in
mechanisms of neurodegeneration (see Section 1.2), the
response of the host brain to infections is a strong reminder
of the functional consequences of inflammatory activity in
non-neurodegenerative pathologies. Indeed, the dysfunction
resulting from alterations of this dialogue is fatal in rabies, for
which no therapy is available when the virus has reached the
CNS, is fatal in HAT if left untreated, and can be fatal in cerebral
malaria even after treatment.
In the context of an issue dedicated to Camillo Golgi, historical
notes on these diseases will be presented, with a focus on Golgi's
influence and legacy in knowledge on rabies and malaria.
Current views on neuroinflammation are first briefly discussed.
1.1. Neuroinflammation: an old concept with
novel implications
Different non-neuronal as well as neuronal cell types in the
nervous system have been observed since the birth of modern
and depicted by Golgi himself by means of the “black
reaction”, his silver impregnation that has provided a key
tool to unravel the structure of the nervous tissue (Fig. 1).
Alterations not only of neurons but also of non-neuronal cells
in disease have been observed since pioneering neuropatho-
logical studies. In particular, as mentioned further, the
description of inflammatory features in brain infections has
provided historical pillars to the neurobiology of disease. A
central role in historical and current knowledge of CNS
inflammation has been played by multiple sclerosis. A wealth
of studies have been dedicated to this disease, a long
recognized example of inflammatory autoimmune CNS pa-
thology, in which blood-derived mononuclear leukocytes are
recruited into the CNS parenchyma (Streit et al., 2004;
Engelhardt, 2010) and that includes a neurodegenerative
component (Glass et al., 2010). Damage in multiple sclerosis
and in its animal model experimental autoimmune enceph-
alomyelitis targets oligodendrocytes, but demyelination and
its pathogenesis are beyond the scope of the present overview.
By addressing the three diseases mentioned above, we will
instead focus on the interplay of astrocytes, microglia, and
leukocyte infiltration (when it occurs) in inflammatory
responses of the brain parenchyma.
1.1.1. Immune specialization versus immune privilege within
the CNS
It is noteworthy, in this context, to recall that the concept of
“immune privilege” within the CNS has been substantially
revised in the last decades (e.g. Galea et al., 2007; Wilson et al.,
2010). Immune privilege is the ability of certain sites in the body
to tolerate the introduction of antigen without eliciting an
inflammatory immune response (Arck et al., 2008). The concept
that the brain parenchyma evades systemic immunological
recognition dates back to the early 1920s (Galea et al., 2007). The
154 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
Fig. 1 – Pyramidal neurons and glia as drawn and stained by
Camillo Golgi. Top: Golgi's drawing of different cell types
impregnated by his staining in the cerebral cortex; the
original figure legend mentions “connective elements”
(referring to glia) disseminated among “ganglion cells”
(neurons). The drawing is part of Table 5 illustrating the
histology of the nervous tissue (Golgi, 1883). Bottom:
pyramidal neurons and glia in the cerebral cortex of the
guinea pig impregnated with the Golgi staining.
Photograph taken from an original preparation of Camillo
Golgi; examination of the slide was kindly permitted by the
Department of Experimental Medicine, Section of General
Pathology “C. Golgi”, of the University of Pavia.
term immune privilege was then coined by Medawar (1948),
who described that immune-mediated inflammation is limited
in certain organs, including the brain, after inoculation of
allotransplants.
Several features of the CNS contribute to its “privileged”
status, including the lack of an obvious lymphatic system
and the paucity or absence of leukocytes patrolling the brain
parenchyma, the low constitutive levels of expression of
major histocompatibility complex (MHC) antigens class I and
II and, importantly, the presence of diffusion barriers and
especially the blood–brain barrier (BBB). The concept of the
existence of this barrier dates back to Paul Ehrlich's
observation that the CNS is not stained after peripheral
injection of water-soluble vital dyes (Ehrlich, 1885). It is now
well established that the main constituents of the BBB are
the cerebral capillary endothelium, perivascular pericytes,
astrocytic end-feet, and their respective basement mem-
branes (Fig. 2A). Endothelial cells, in particular, are joined by
tight junctions that force most macromolecular traffic across
the BBB through a transcellular route. As also illustrated in
the paper by Verkhratsky et al. (in press) in this issue, the
peculiar relationship between astrocyte end-feet and blood
vessel walls had already been clearly depicted by Golgi. The
close apposition of astrocytic end-feet processes to capillar-
ies creates an interface through which astrocytes, the most
abundant glial cell type in the CNS and major components of
the neurovascular units, can monitor and influence blood
supply and vascular activity (Abbott et al., 2006).
1.1.2. Activation of astrocytes and microglia in the
immune response
Astrocytes and their role in synaptic neurotransmission, as
well as microglia in the healthy brain and in disease, are the
subject of other review articles in this issue (Grafstein, 2010;
Matteoli and Verderio, 2010; Verkhratsky et al., 2010). It is here
only recalled that microglia, cells of the myeloid lineage, are
considered the CNS resident macrophages, albeit with a
downregulated immunophenotype compared with peripheral
macrophages, i.e., adapted to the CNS microenvironment
(Galea et al., 2007). Microglia are now known to represent the
main cell type of the CNS innate immune system. Upon all
kinds of challenges and injury, microglia are alerted and
undergo a rapid, temporally and spatially regulated process of
activation, which includes antigen presentation, state shifts,
and changes of molecular repertoire, reaching the state of a
phagocytic element (Hanisch and Kettenmann, 2007; Rivest,
2009). Activated microglia can thus generate inflammatory
mediators, including proinflammatory cytokines, free radicals
and complement, which in turn induce chemokines and
adhesion molecules, recruit immune cells, and activate other
glial cells. Activated microglia can also generate anti-inflam-
matory molecules. Therefore, microglia activation can have
both detrimental and beneficial effects, whose relative impact
is currently highly debated. In particular, in line with the
concept of functional polarization of macrophages into M1
and M2 cells (Mantovani et al., 2004), “classical activation” of
microglia (M1) can exert cytotoxic effects through to the
release of reactive oxygen species and proinflammatory
mediators, while “alternative activation” of microglia (M2)
has been implicated in protective effects on endangered
neurons through the release of trophic factors and anti-
inflammatory cytokines and/or chemokines (Carson et al.,
2006; Block et al., 2007; Glezer et al., 2007; Verkhratsky et al.,
2010). It should also be considered that phagocytic clearance
by microglia following injury plays an important role in
facilitating the reorganization of neuronal circuits (Neumann
et al., 2009; Walter and Neumann, 2009). As protagonist of the
brain immune surveillance, microglia exert a central role also
in the defense against infectious agents in the CNS. This
function, however, has been mainly investigated in the
context of the human immunodeficiency virus-related ac-
quired immunodeficiency syndrome (HIV/AIDS), in which
microglia is the main cell type responsible for CNS infection,
leaving open many questions regarding other infectious
diseases of the brain (Rock et al., 2004).
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3 155

Fig. 2 – Schematic representation of histopathological correlates of brain infections (rabies in B, cerebral malaria in C, human
African trypanosomiasis in D), compared to the normal organization (shown in A) of the brain parenchyma and of the
blood–brain barrier (BBB). The normal brain parenchyma (A) is represented by a single neuron (pale yellow), astrocytes (orange),
and microglia (blue); oligodendrocytes and perivascular macrophages are not shown. Astrocytic end-feet contribute to the
formation of the highly selective BBB, together with the blood vessel's tightly bound endothelial cells and their respective
basement membranes (represented for simplicity as a single green band). During rabies infection (B), monocytes cross the BBB as
a consequence of a chemokine-cytokine gradient and migrate into the parenchyma. Infected neurons upregulate the expression
of membrane proteins that are recognized by corresponding receptors on the T cell surface. The interaction between neurons
and infiltrated lymphocytes triggers exhaustion and/or death of CD3/CD8+ T cells. Axonal transport and transneuronal spread of
the rabies virus occur without apparent disruption of the affected neuronal networks. Notice the inclusions (Negri bodies) in the
cytoplasm of the infected neuron, characteristic of rabies virus infection (see also Fig. 4). The response of parenchymal glial cells
is in general mild in experimental and human rabies. A central event of cerebral malaria (C) is cerebral sequestration of
erythrocytes infected by Plasmodium falciparum, with adherence of parasitized red blood cells among themselves (agglutination),
to the endothelial wall, and to non-parasitized erythrocytes (rosetting). These cellular aggregates cause clogging of blood vessels
of small caliber, as well as various degrees of damage to the blood vessel wall, leading also to hemorrhages (as depicted in the
figure) and petechiae. Changes in astrocytes and especially of microglia have been observed in human cerebral malaria and in its
murine model, but marked glial cell activation does not seem a distinctive feature of cerebral malaria. In human African
trypanosomiasis (D), entry of the parasite Trypanosoma brucei into the brain parenchyma across the blood–brain barrier marks
the progression from the hemolymphatic stage to the second, meningoencephalitic stage of the disease. Experimental findings
have shown this event occurs together with recruitment of lymphocytes to the brain parenchyma. After crossing the
endothelium, trypanosomes may remain “on hold”, confined within the two basement membranes and appearing as “cuffing”
the vessel in histological sections (not shown here) before migrating into the parenchyma. Activation of astrocytes and microglia,
which may show regional prevalence, is part of the pathology of human African trypanosomiasis.
156 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
Astrocytes, which are of neuroectodermal origin, also
undergo upon insults a process of activation. This leads to
molecular, phenotypic, and functional changes that can alter
astrocyte activity through both gain and loss of function.
Astrocyte activation occurs along with a fine, gradual pro-
gression of changes that influence both neighboring neurons
and other non-neuronal cells (Sofroniew and Vinters, 2010).
Activated astrocytes are endowed, as microglia, with the
ability to secrete cytokines and chemokines, which exert an
impact on both adaptive and innate immune responses
(Farina et al., 2007). Astrocytes express receptors, including
Toll-like receptors (TLRs), which are also expressed by
microglia and neurons and represent key molecules in the
innate immune response (Okun et al., 2009).
1.1.3. The growing interest in the neuroinflammatory
component of neurodegenerative diseases
Due to a wealth of exciting new molecular and cellular data,
neuroinflammation stands now as a central event of CNS
disease. In particular, as it will be outlined below, neuroin-
flammation is currently closely associated with chronic
neurodegenerative diseases. Interest in the impact of neu-
roinflammation on brain infections seems instead rather
limited.
Progress of knowledge and its focus is also demonstrated
by the trends in the publication of articles. A paper which
appeared in July 2004 stated that a PubMed search using
“neuroinflammation” as the only key word yield about 300
papers, none before 1995 (Streit et al., 2004). This number has
increased more than 6 times in 6 years, as in September 2010
the same search yields about 1950 papers. Using the key words
“glia” and “Alzheimer's disease” yields about 2950 papers
(“microglia” and “Alzheimer's disease”: about 1750 papers);
“glia” and “cerebral malaria” yields 25 papers (“microglia” and
“cerebral malaria”: 16 papers).
The fact that neuroinflammation is now regarded as a
key component of neurodegenerative disease is also based
on novel findings on the participation of multiple cell
types to pathogenetic mechanisms of neurodegeneration.
This is briefly discussed below to set the stage of current
perspectives.
1.1.4. The concept of “non-cell autonomous”
neurodegenerative disease
Glial cell changes have been observed in Alzheimer's disease
since its first description (Kettenmann and Verkhratsky,
2008; Verkhratsky et al., 2010). However, many neuropatho-
logical conditions including all major neurodegenerative
diseases, such as Alzheimer's disease, Parkinson's disease,
Huntington's disease, and amyotrophic lateral sclerosis
(ALS), have traditionally been considered, rather mechanis-
tically, “cell autonomous”, meaning that damage affecting a
discrete population of neurons is sufficient to produce
disease. This concept stems from the pivotal idea, estab-
lished in the 19th century as a legacy of the cell theory first,
and of the neuronal doctrine much later, that individual
cells function autonomously, while being part of the whole
organism.
A drastic revision of this concept in the CNS is very recent
history. “Non-cell-autonomous” events, i.e., those requiring
the participation of multiple cell types, are widely documen-
ted in biology (e.g. developmental processes) and pathology
(e.g. tumor growth). A PubMed search indicates that the
terminology of “non-cell-autonomy” started to be adopted at
the end of the 1980s to describe gene function in Drosophila
(Vinson and Adler, 1987), and cytokine effects on tumor cell
types in mice (Tepper et al., 1989). Soon after, it was reported
that defective glia may contribute to neurodegeneration in a
mutant of Drosophila (Buchanan and Benzer, 1993).
Evidence that non-neuronal cells are main players in the
pathogenesis of neurodegenerative diseases in mammals is
even more recent and derives primarily from experimental
studies on ALS. This fatal disease is notably characterized by
the loss of upper (cortical) and lower (cranial and spinal)
motoneurons; about 10% of the ALS cases are familial (FALS),
with pathological features similar to those of the more
common sporadic form. A missense mutation in the gene
encoding the enzyme Cu/Zn superoxide dismutase 1 (SOD1)
was discovered in 1993 in ALS patients; this led the following
year to the creation of a transgenic mouse strain which
overexpresses the human mutant SOD1 gene and exhibits a
phenotype with many features in common with ALS (Gurney,
1994). Investigation on the pathogenetic mechanisms of FALS
in this murine model developed rapidly. Increasing astrocyte
activation with disease progression was demonstrated,
implicating possible interactions between motoneurons and
non-neuronal cells in the disease (Brujin et al., 1997; Levine
et al., 1999). The study of the effect of caspase inhibition in
FALS mice pointed to a non-cell-autonomous regulation of
the regulatory pathway of caspase expression (Li et al., 2000).
Also, data indicating that accumulation of mutant SOD1 in
motoneurons may not be sufficient or critical to induce or
accelerate disease in FALS mice led to the hypothesis that
the pathogenic processes may involve non-neuronal cells
(Lino et al., 2002).
Results obtained in chimeric mice showed that neurode-
generation is delayed or absent when mutant SOD1-expres-
sing motoneurons are surrounded by healthy wild-type cells
and, vice versa, when wild-type motoneurons are surrounded
by mutant SOD1-expressing cells (Clement et al., 2003). It was
then demonstrated that free radicals released from activated
microglia may initiate motoneuron injury by increasing the
susceptibility of motoneuron glutamate receptor to the toxic
effects of glutamate (Zhao et al., 2004).
Based on these and other findings, a wealth of evidence
from diverse approaches has converged to demonstrate that
in murine FALS motoneuron damage is enhanced by the
inflammatory response of non-neuronal neighboring cells,
which accelerates disease progression (Boillée et al., 2006).
This is also raising interest in view of novel therapeutical
approaches. While experimental models as close to the
human pathology as the SOD1 mice are not yet available for
most other neurodegenerative diseases, this concept is now
being extended to Parkinson's, Alzheimer's, and Hunting-
ton's diseases and other neurodegenerative diseases as well
(Garden and La Spada, 2008; Ilieva et al., 2009; Hirsch and
Hunot, 2009; Glass et al., 2010). Used and abused, the
definition of “non-cell autonomous” event is thus increas-
ingly used by investigators who focus on neurodegenerative
processes.
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
As it often happens with successful terminology, the
neuroscience community has adopted the concept of “non-
cell autonomous disease” regarding it as novel. While there
is indeed novelty in the recognition of the importance of
non-neuronal cells in the pathogenesis of neurodegenera-
tion, it should be remembered that the crucial role of the
interaction between cell types in infectious diseases of the
nervous system has been known for decades. It is well
established that non-neuronal cells contribute to and, in
some instances, represent the primary cause of profound
disturbances of neural function, whether or not overt
neuronal damage can be detected at the histopathological
investigation. The infectious diseases dealt with below
provide striking examples of such “non-cell autonomous”
CNS pathology.
2. The fight of rabies virus against
neuroinflammation: Evading the
immune response
2.1. The discovery of Negri bodies
Rabies has been known to humankind throughout its history,
as documented by early writings on the fatal consequences of
bites by rabid dogs. For example, the code of laws from the
Mesopotamian city state of Eshnunna (19th–18th centuries
BC) imposed monetary fines on the owner of a rabid dog that
had bitten a citizen causing death, with a discount if the dog
bite had caused the death of a slave (Fales, 2010). Early
descriptions of rabies from China date back more than
2500 years (Hu et al., 2009).
The milestone in the history of rabies is represented by the
well-known development by Louis Pasteur (1822–1895) of the
first rabies virus vaccine from the spinal cord of rabid rabbits.
Pasteur continued and extended studies carried out by Pierre-
Victor Galtier (1846–1908) who had used the rabbit as
experimental animal in rabies research and had shown for
the first time the transfer of rabies from one animal to
another (Galtier, 1879). Pasteur's vaccine was based on the
inoculation of rabies-infected material, and the first patient
was the 9-year-old Alsatian boy Joseph Meister. In October
1885, Pasteur addressed the Academy of Sciences in Paris to
announce his results, declaring that the boy had “escaped not
only the rabies that he might have received from his bites, but
also the rabies that I inoculated into him” (Pasteur, 1885).
The diagnosis of rabies remained, however, a serious
problem, which many investigators were engaged to solve.
Pasteur's vaccination had created the need for a diagnostic
method that could rapidly demonstrate the presence of the
disease in the animal in order to decide whether to vaccinate
the bitten person. The search for characteristic patho-
logical features of rabies was, therefore, a priority. In a
seminal study on the neuropathology of rabid animals, Victor
Babes (1854–1926) described a number of alterations but
concluded that brain lesions in rabies do not exhibit
characteristic features (Babes, 1892). He did describe, howev-
er, aggregates of cells with immature, “embryonic” appear-
ance, which he named “rabies nodules”. These aggregates
157
could represent microglial nodules; however, in the drawings
which illustrate Babes' (1892) article, they actually appear as
aggregates of lymphocytes.
Camillo Golgi, a skilled physician, was very interested in
diseases and in the experimental approach to clinical pro-
blems (Mazzarello, 2010). Like other investigators of his time,
Golgi was especially interested in infectious diseases. In 1886,
he studied the brain of a human victim of rabies but did not
observe specific neuropathological features (Mazzarello, 2010).
He then assigned to his assistant Adelchi Negri (1876–1912)
(Fig. 3) the task to investigate the histopathology of rabies,
searching for the aetiology of the disease. Negri had been an
intern student in Golgi's laboratory during his medical studies
at the University of Pavia and had obtained his medical degree
in 1900.
Negri worked very hard and infected experimental dogs
and rabbits with the rabies street virus (see Section 2.3.1). At
the histological study, Negri could observe characteristic
corpuscles in nerve cells of the brain and spinal ganglia and
presented his results in March 1903 at the “Società Medico-
Chirurgica” (Medical and Surgical Society) of Pavia. In his
communication, Negri (1903) stated that the corpuscles could
be seen with different histological stains, including hema-
toxylin–eosin (provided the staining was not too intense), and
that he had obtained the best results with the methyl blue
eosin method of Mann, with which the corpuscles exhibited a
reddish color (Fig. 4). He provided a detailed description of the
appearance and size of the intracellular corpuscles, which
were mostly located in the cytoplasm and could invade the
dendrite stem. The corpuscles showed a preferential locali-
zation at certain sites, including the Purkinje cells of the
cerebellum and the pyramidal cells of the Ammon's horn of
the hippocampus. They could also be observed in the
Ammon horn in fresh tissue and in unstained sections,
which demonstrated that they were not staining artifacts and
also had obvious practical implications. Negri clearly stated
his firm belief that the corpuscles were specific features of
rabies. He also stated that the corpuscles could represent a
product of degenerative processes of cell constituents. He was
convinced, however, that the corpuscles corresponded to
different steps of the developmental cycle of a protozoan that
caused the disease. In his interpretation, Negri was probably
reinforced by Golgi's studies on the malaria parasite (see
Section 3.2).
Some months after Negri's discovery of intraneuronal
inclusion bodies in rabies, Alfonso di Vestea in Naples and
Paul Remlinger and Riffat Bey in Costantinople showed that
the etiological agent of the disease was a filterable virus.
However, Negri tried until 1909 to demonstrate that the
different sizes and structures of the intraneuronal bodies
named after him corresponded to different steps of the
developmental cycle of a protozoan (Bentivoglio, 2003;
Margreth, 2003).
Also Santiago Ramón y Cajal engaged in the task of
searching for neuronal changes that could be diagnostic of
rabies. “I hoped to discover some variation more or less
typical of infective processes in the nervous system that
could be utilized in diagnosis” wrote Cajal (1996) in his
memoirs. He investigated the brain of rabies-infected ani-
mals in a study in which he was “zealously assisted by
158 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3

Fig. 3 – Portraits of two pioneers of studies on rabies. Left: Adelchi Negri (1876–1912), who discovered the intraneuronal
inclusions (Negri bodies) pathognomonic of rabies infection. Right: Ernesto Bertarelli (1873–1957), who discovered the
centrifugal spread of the rabies virus to the salivary glands in rabid animals.

D. Dalmacio Garcia, chief of the Veterinary Section of the
National Institute of Hygiene”. In his study Cajal stated that
Negri bodies were likely to represent degenerative products of
the neuronal cytoplasm and devoted attention to nerve
constituents, and in particular neurofibrils (Cajal and García,
1904). Cajal detected hypertrophy of neurofibrils and vacu-
olar damage of some neurons but does not seem to have
identified changes specific of rabies. His slides of rabies-
infected brains are kept at the Cajal Museum in Madrid
(Garcia-Lopez et al., 2010).
Independently of his incorrect etiological hypothesis,
Negri's findings provided a breakthrough in the diagnosis of
rabies. At that time, the diagnosis was based on a biological
test that took 2 or 3 weeks to complete, and suspected
animals were confined to observe whether they developed
the disease. The detection of Negri bodies in neurons allowed
instead the rapid identification of rabid animals by exami-
nation of brain tissue. Negri's wife and coworker, Lina
Luzzani, who published after his death an account of
his work on rabies stressing its practical implications,

Fig. 4 – Negri bodies, pathognomonic of rabies infection of the central nervous system, and the time of their discovery and today.
Left: Drawing of Adelchi Negri of the intraneuronal inclusions he observed in the cytoplasm of rabies-infected neurons of a dog
after subdural inoculation with rabies street virus; Mann staining (from Negri, 1903). Right: Schematic model of Negri bodies
according to the findings (reported by Ménager et al., 2009) that these inclusions are formed by cytosolic aggregates of Toll-like
receptor 3 (TLR3) coated by viral nucleocapsid (NC) proteins. Nu, neuronal nucleus.
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
emphasized that the detection of Negri bodies allowed the
diagnosis within a few hours “after receiving the animal's
head” (Negri Luzzani, 1913).
The biological significance of Negri bodies remained,
however, enigmatic and continued to be debated for decades,
even when new knowledge on intraneuronal inclusions in
other infections as well as in non-infectious diseases started
accumulating (Kristensson et al., 1996; Bentivoglio, 2003).
2.2. The discovery of centrifugal spread of rabies virus to
the salivary glands
Negri's experimental investigations on rabies raised the
interest, among others, of Ernesto Bertarelli (1873–1957)
(Fig. 3). Bertarelli, who graduated at the University of Turin
in 1898, was obviously very influenced by Golgi's experimental
approach to diseases. He was then professor of Hygiene at the
University of Pavia (the same University where Golgi had been
professor) from 1919 to 1946, and his esteem of Golgi is
witnessed by a celebration he wrote on the occasion of the
25th anniversary of Golgi's death (Bertarelli, 1950).
At the time of Negri's report in March 1903, Bertarelli was
assistant professor at the Institute of Hygiene of the University
of Turin. Obviously thrilled by Negri's discovery, Bertarelli,
together with his coworker Guido Volpino, investigated the
brain of a victim of rabies deceased in May 1903. After less
than 1 month, he reported his findings at the Medical
Academy of Turin. This study (Bertarelli and Volpino, 1903)
provided the first report of Negri bodies in the CNS of the
human brain infected by rabies. Numerous Negri bodies were
found in the Ammon horn and in Purkinje cells of the
cerebellum. Furthermore the observations raised doubts on
the possibility that Negri bodies could represent a parasite,
and Bertarelli worked in subsequent studies at confirming
that rabies was due to a filterable agent.
Interested in the routes of spread of the rabies causative
agent, Bertarelli then tested experimentally the hypothesis of
spread of the “rabies virus” through blood vessels, lymphatic
vessels, or nerves. After cutting vessels or nerves serving the
salivary glands of rabbits prior to inoculation with material
from dogs infected with the street rabies virus, Bertarelli
(1904a,b) reported that “the only route through which the
virus reaches the gland is the nerve”. This study is included,
together with that of Negri, among the notable milestones in
the history of rabies in the 20th century (Rupprecht et al.,
2002). Centrifugal spread of rabies virus to the salivary gland,
skin, cornea, and other peripheral organs is now part of
classical knowledge on rabies pathogenesis and transmission
(Jackson, 2008). Notably, Bertarelli's findings pioneered sub-
sequent discoveries on axonal transport as a cardinal
function of neurons, exploited by toxins and infectious agents
in pathological conditions (Bentivoglio, 1999).
Bertarelli (1906a,b) concentrated then on syphilis and was
the first to succeed in the transmission of the infection to
laboratory animals, and in particular to rabbits. This repre-
sents a milestone in the studies on syphilis, which had been
considered an exclusively human disease, and Bertarelli's
experiments had been preceded by (unfortunately successful)
inoculation of Treponema in humans and unsuccessful
inoculation in monkeys (Sherwood, 1999).
159
2.3. Rabies today
As recently emphasized (Wunner and Briggs, 2010), it is
“astonishing” that 50,000 to 55,000 people die from rabies
worldwide each year, and that over 3 billion people are at risk
of rabies virus infection in over 100 countries. Over 95% of the
victims are reported in Asia (with 25,000–30,000 victims in
India alone) and Africa, where canine rabies is far from being
eliminated, and nearly all are victims of a rabid dog bite; a
large proportion of them are children. The victims are
probably under-reported, and deaths due to rabies are
responsible for 1.4 million disability-adjusted life years
(DALYs) in Asia and Africa (Knobel et al., 2005). Rabies has
been reemerging in China in recent years, raising public
concern (Hu et al., 2009; Song et al., 2009). A concern was raised
even by officials of the 2010 FIFA World Cup in South Africa;
visitors were informed about rabies and rabies prophylaxis
before or after an exposure to a potentially rabid animal
(Malerczyk et al., 2010).
While all mammals are susceptible to and capable of
transmitting the rabies virus, carnivorous mammals and bats
serve as major hosts and reservoir of the virus (Rupprecht
et al., 2002; Nigg and Walker, 2009). The dog is responsible for
the majority of human cases, especially in developing
countries. Other major wild carnivorous species that are
rabies virus reservoirs include various species of foxes from
all continents. Oral vaccination programs involving aerial
bait distribution has been successful in rabies control in
foxes in Europe and North America and in coyotes in Texas.
Vaccination programs against raccoon rabies have also been
launched in North America. An alarm has been raised very
recently in Northeastern regions of Italy by the reemergence
of rabies (which had been eradicated in the mid-1970s) in
foxes crossing the border with Slovenia, and an aerial oral
fox vaccination program has been implemented in the
winter 2009–2010 (Capello et al., 2010). Bat rabies viruses
are responsible for most of the few human cases in high-
income countries where the prevalence of domestic animal
vaccination is high. In North America and Germany there
have been in 2004 unfortunate cases of human rabies
transmitted to recipients of organ transplantation from
unsuspected rabid donors, all resulting in the death of
organ recipients.
2.3.1. Rabies virus transmission and clinical features of
the infection
Rabies is caused by neurotropic RNA viruses (family Rhabdo-
viridae; genus Lyssavirus). Lyssaviruses are a collection of
genetically related viruses, adapted to replication in the
mammalian nervous system. Rabies viruses include 11
genotypes, of which genotype 1 (including the classical rabies
virus) is the most prevalent (Schnell et al., 2010). There are
two general strains of rabies virus: street virus and fixed virus.
Street virus comes directly from the CNS of a naturally
infected host (i.e., it has been obtained in the laboratory after
primary isolation in animals) and has undergone minimal or
no adaptation in experimental animals or cell cultures. The
incubation period and biological behavior of the street virus
are variable. Fixed virus has instead been adapted through
passages in the brain of experimental animals and/or cell
160 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
culture, so that the incubation period and biological behavior
have become “fixed”.
The rabies virus life cycle is highly regulated at every step
(Schnell et al., 2010). The virus first binds to the host cell
receptor/s and enters the host cell by endocytosis; next, the
endosome containing the virus is transported retrogradely
through the axon of the infected neuron. Once in the cell body,
the fusion of the viral and endosomal membranes releases the
viral genome in the cytosol (“uncoating”). Viral components
are here produced through transcription, replication, and
protein synthesis and subsequently assembled and trans-
ported to the site of budding. Thus, mature rabies virions are
released and can start a new round of infection.
Data on rabies transmission and clinical features have
been presented in recent reviews (Rupprecht et al., 2002;
Hemachudha et al., 2002; Jackson, 2008; Nigg and Walker,
2009). Human transmission of the rabies virus occurs primar-
ily through bites of rabid animals, where infected saliva
penetrates the skin and the virus enters the nervous system
via motor nerves, through the neuromuscular junction, or
sensory nerves.
A specific receptor for rabies virus has not yet been
identified (Rupprecht et al., 2002; Schnell et al., 2010). The
virus glycoprotein is required for the first step of attachment
to the host neuron, but host cell molecule/s interacting with
the rabies virus glycoprotein to mediate its entry remain to
be defined. Interaction of rabies virus with postsynaptic
nicotinic acetylcholine receptors at the neuromuscular
junction could enable efficient infection and be instrumental
in transferring the virus from the periphery to the CNS, but
receptor/s on the presynaptic nerve membrane should be
used for the initial entry in motoneurons. The p75 low-
affinity nerve growth factor receptor has been reported to
represent a potential rabies virus receptor. However, the
uptake and transport of rabies by a wide variety of neuronal
cell types suggest that receptors for the virus are ubiquitous,
and different receptors (and, potentially, co-receptor mole-
cule/s) may be used. To date, a strong candidate is the
neuronal cell adhesion molecule (NCAM) because of its
presynaptic localization and its widespread distribution in
the nervous tissue (Lafon, 2005).
The incubation period of rabies is the most variable among
viral infections of the CNS; typically is 1–2 months, but the
range is from a few days to several years. A prodromal stage,
lasting a few days to 2 weeks, begins once the virus has
travelled to the dorsal root ganglia and CNS. Symptoms are
variable and non-specific, such as headache, malaise, irrita-
bility, and nausea. Invasive replication of the virus in the CNS
leads to the acute neurologic phase.
The clinical picture of rabies has been defined as “horri-
fying” (Hemachudha et al, 2002). The disease can manifest as
encephalitic (furious) rabies, representing approximately
two-thirds of the cases, or paralytic (dumb) rabies. The
determinants for the two types of clinical rabies are still
discussed, and they seem to depend on many factors related
to the virus and to the host. It has even been reported that the
same dog has caused furious rabies in one human victim and
paralytic rabies in another (Hemachudha et al., 1988).
Symptoms of encephalitic rabies include irritability, hyper-
activity, hallucinations, hypersalivation, hydrophobia (fear of
water), and aerophobia (fear of air), resulting from painful
laryngeal and pharyngeal spasms. Fluctuating conscious-
ness, with periods of agitation and depression, phobic or
inspiratory spasms and autonomic dysregulation represent
cardinal symptoms. In paralytic rabies, flaccid quadriplegia
occurs and initiates symmetrically or asymmetrically. Coma
occurs in both forms in the late stage of clinical progression,
invariably followed by respiratory failure and death within a
few days, for the encephalitic form, or a few weeks, in the
paralytic form.
The direct fluorescent antibody (DFA) test, developed
during the late 1950s, is currently used for rabies diagnosis,
and RT-PCR and other molecular assays can be useful as
confirmatory test, but have limited benefits as routine
procedure (Rupprecht et al., 2002). As presented previously,
before the development of the DFA, the diagnosis was based
on the identification of Negri bodies in brain tissue from
animals with suspected rabies. This is still in use in developing
countries where immunofluorescence facilities may not be
available (Kashyap et al., 2004).
2.3.2. Neuropathology of rabies
The most puzzling aspect of rabies pathology, both in human
and experimental infections, is the contrast between the very
severe clinical picture and the relatively mild neuroinflam-
matory signs and overall neuronal sparing, in spite of the
pathognomonic presence of Negri bodies. In other words,
based on the neuropathology, it is far from obvious what the
causes of the very severe neurological symptoms may be.
The preferential localization of Negri bodies in sites such as
the hippocampus has been confirmed in modern studies
(Rupprecht et al., 2002; Suja et al., 2009) and has been
implicated in the behavioral disturbances which lead to
extreme aggressiveness during the disease, strategic for
virus transmission through bites. However, localization of
the virus at other brain sites, and especially brain stem and
thalamus (Bentivoglio and Kristensson, 2004; Suja et al., 2009),
is also likely to be very relevant in terms of behavioral
dysfunction.
In human victims, lymphocytic infiltration has been
reported especially in the spinal nerve roots and dorsal root
ganglia in paralytic rabies, with milder degree of inflamma-
tion in furious rabies. In the spinal cord, inflammatory cell
infiltration around small blood vessels (perivascular cuffing)
and microglia proliferation have been observed in both
paralytic and furious rabies, but such inflammatory features
can also be very mild (Mitrabhakdi et al., 2005), and absence of
gliosis or macrophage infiltration, with no neuronal apoptotic
changes, has also been observed (Tobiume et al., 2009).
Damage of ventral horn neurons has, however, been observed
in paralytic rabies, but such finding is inconsistent and
ventral horn neurons can be also be intact (Mitrabhakdi
et al., 2005).
Also experimental studies have pointed to the absence of
overt neurodegeneration after rabies virus infection, though
subtle changes may occur. In an early investigation of the
hippocampus of mice experimentally infected with street
rabies virus, neurons appeared well preserved except for
the presence of Negri bodies (Miyamoto and Matsumoto,
1967). In a recent study of transgenic mice expressing yellow
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
fluorescent protein in neuronal subpopulations infected with
a strain of fixed rabies virus, histopathological changes were
minimal, but fluorescence microscopy analyses revealed
beading and fragmentation of dendrites and axons in
neuronal subsets and vacuolation in pyramidal neurons of
the cerebral cortex and hippocampus corresponding to
swollen mitochondria at the electron microscopic level
(Scott et al., 2008).
Consistently with the findings reported above, the brains
of dogs infected by the street virus exhibit, especially in the
grey matter, a variable, sometimes minimal degree of
inflammation, with microglia proliferation and a very
widespread distribution of viral nucleocapsid protein (Suja
et al., 2009). Neurons, however, do not show major altera-
tions, and even those infected with a heavy viral load have an
intact nucleus and nucleolus (Suja et al., 2009). Magnetic
resonance imaging and virological analyses, as well as
analyses of the expression of interleukin (IL)-1β and inter-
feron (IFN)-γ transcripts, have shown that inflammation is
more moderate in furious dog rabies and the virus more
neuroinvasive at an early stage than in paralytic rabies
(Laothamatas et al., 2008).
It is interesting to note that, at variance with the lack of
apoptotic changes in neurons, some lymphocytes, perivascular
macrophages and microglia, and endothelial cells were
observed to undergo apoptosis in the brain of rabid dogs
(Suja et al., 2009; Schnell et al., 2010). The spread of
pathogenic rabies virus through neuronal networks requires
the integrity of the network, so that the virus progression is
not interrupted by destruction of the infected neurons.
Rather, the virus seems to trigger the death of non-neuronal
cells, which represents, at least in part, a mechanism to evade
the host brain immune response, as presented below (see
Section 2.2.3).
Drastic inhibition of the synthesis of proteins required in
maintaining neuronal functions has been implicated, howev-
er, in the lethal effect of rabies virus infection (Dietzschold
et al., 2005). In fact, the study of overall expression profiles of
host genes in the infected mouse brain has revealed that the
predominant effect of rabies virus infection is the down-
regulation of gene expression (Prosniak et al., 2001). The same
study, on the other hand, has reported the activation in the
brain of a number of host genes which may be involved in the
replication and spread of rabies virus.
Although rabies virus replication in the CNS occurs
primarily in neurons, glial cells can be infected by the
virus, as shown by in vitro and in vivo evidence of viral gene
expression in astrocytes and microglia (Ray et al., 1997;
Nakamichi et al., 2005; Suja et al., 2009). In particular, in vitro
findings have indicated that virus-encoded proteins can be
expressed in the cytoplasm of microglial cells, in which,
however, the synthesis of viral genome and production of
virus progenies are significantly impaired compared to
neurons (Nakamichi et al., 2005). Furthermore, the expres-
sion of the chemokines CXCL10 and CCL5 is induced in
microglia infected by rabies virus, and such induction is
regulated by multiple signaling pathways activated by the
infection (Nakamichi et al., 2005). Although the significance
of such findings in rabies pathogenesis remains to be
elucidated, they indicate that rabies virus activates host
161
genes in microglia, at variance with the findings observed in
neurons. These data also implicate chemokine release by
microglia in leukocyte recruitment during the infection. On
the other hand, studies with laboratory-attenuated rabies
virus have shown that the balance and role of chemokine
induction is an important mechanism in controlling rabies
virus infection but is also a multifaceted response which
may be detrimental for the host (Kuang et al., 2009; Zhao
et al., 2009).
2.3.3. Negri bodies today and evasion of the immune response
in rabies
Knowledge of Negri bodies has been recently enriched by
novel data. These inclusions contain ubiquitinylated proteins
and the chaperone heat shock protein 70, reminiscent of the
composition of inclusion bodies and/or aggresomes which are
seen in several neurodegenerative disorders (Bentivoglio,
2003). Negri bodies, however, are not aggresomes containing
misfolded protein aggregates but are likely to represent “viral
factories”: they contain all viral RNAs and data indicate that
they represent functional structures for viral transcription and
replication (Lahaye et al., 2009).
Interestingly, an inner core containing a TLR3 aggregate,
surrounded by a viral nucleocapsid protein cage, has been
described in Negri bodies (Ménager et al., 2009) (Fig. 4). TLRs
allow brain cells to recognize and respond to the presence of
danger signals and pathogen-associated molecular patterns
encoded by pathogens. It has been suggested that the
sequestration of TLR3 inside Negri bodies could reduce the
cellular innate immune response and represent the product of
rabies virus strategy to hijack the normal functions of
neuronal proteins favoring the progression of its life cycle
(Ménager et al., 2009). The potential relevance of this recent
observation awaits further clarification.
Mechanisms of immune evasion of the host during rabies
infection also include T cell inactivation. As mentioned
previously, rabies virus infection triggers the production of
chemokines and inflammatory cytokines, leading to the
infiltration of lymphocytes into the brain parenchyma
(Fig. 2B); macrophages are also recruited. Experimental
mouse models have demonstrated that migratory T cells
can control rabies virus infection and that the severity of the
infection is inversely correlated with the number of infiltrat-
ed CD3+ and CD8+ T cells (Lafon, 2008). However, rabies
virus has developed strategies to hamper the action of T cells,
destroying them shortly after their entry into the CNS
(Fig. 2B). Thus, in acute infection T cells decline after their
recruitment to the brain parenchyma, concomitantly with
apoptosis of infiltrating T cells (Lafon, 2008). This is achieved
exploiting immunosubversive proteins which are upregu-
lated by the infection in neurons. Acute rabies induces in
neurons the expression of B7-H1, HLA-G, and FasL proteins,
which inhibit T cell proliferation and cytokine production
and drives death of migratory T cells. At the cell surface of
infected neurons, these molecules interact with the corre-
sponding receptor (Fas for FasL, PD-1 for BH7-H1, CD8 for HLA-
G) expressed by T cells. The binding reduces cell expansion
and promotes active elimination of CD3/CD8+ T cells that
would be protective against the infection, thus favoring viral
invasion (Lafon, 2008).
162 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
These pathogenetic features indicate that rabies virus has
developed sophisticated intracellular and intercellular
mechanisms to evade the host immune response. Through
these mechanisms, rabies virus has a suppressive effect on
the CNS inflammatory response and seems thus to have
evolved a strategy to silence the dialogue between neurons
and non-neuronal, both resident and infiltrating, cells.
2.4. Rabies virus as tract tracing tool
Dealing with the neurobiology of rabies virus infection, it is
relevant to recall that the transsynaptic journey of the virus
can be used for the study of neural circuits. The highly
selective mechanisms of spread of the rabies virus from
neuron to neuron have made it possible to employ the
experimental infection as a powerful tool for neuroanatom-
ical tracing of CNS circuits. In conventional tract tracing, a
substance injected in a carefully mapped location is taken up
by local neuronal membranes and transported along the
axon, thus reaching the neuronal cell body, in the case of
retrograde tracers, or the axon terminals, in the case of
anterograde tracers. Histological labeling of the tracing
substance allows the identification of, respectively, the
location of neurons with terminals reaching the injected
site or the territories innervated by neurons located at the
injection site. While this approach has been instrumental in
modern understanding of brain circuitry, the technique is
very limited in accomplishing the so-called transneuronal
tracing, i.e., the labeling of synaptically connected chains of
neurons. Conventional tracers accumulate inside the neuron,
but only a small amount crosses the cell membrane, and even
less is taken up by adjacent neurons. Thus, injection of a
retrograde tracer near axon terminals results in intense
labeling of the cell body and, at best, very weak labeling of
second-order neurons.
The use of neurotropic viruses as neuroanatomical tracers
elegantly addresses this issue. Inoculated viruses infect
neurons and are transported to the cell body where they
replicate; newly generated viruses exit the cell and infect
second-order neurons, where the process is repeated. Two
main classes of viruses are currently employed in the
laboratory: the alpha-herpesviruses (herpes simplex virus
type 1 and pseudorabies) and the rabies virus (Ugolini, in
press). The latter has proven particularly useful, thanks to
several specific features. First, the “fixed” strains of rabies
virus, i.e., viruses specifically adapted for laboratory use (see
Section 2.3.1), only propagate retrogradely; it has been
demonstrated, in fact, that the virus is actively transported
in the axon towards the cell body, and that newly formed
(fixed) viruses spread from the soma to the dendrites, but do
not enter the axon (Ugolini, 1995). Second, viruses exit the
cell via membrane budding (Schnell et al, 2010), leaving the
neuron intact and therefore available for histological label-
ing. Third, binding of the virus to higher-order neuronal
membranes and subsequent internalization seems to occur
in the proximity of presynaptic terminals. Thus, rabies virus
tracing highlights functional connectivity rather than mere
contiguity. In this respect, it is crucial to identify the
neuronal membrane receptor for the rabies virus (see Section
2.3.1). Taken together, the unidirectional retrograde transfer
of fixed rabies virus, the infection of new neurons occurring
near the synapses, the survival of affected neurons, and the
fact that the infection can be traced along an arbitrary
number of trans-neuronal “jumps” make the experimental
rabies infection an invaluable neuroanatomical tool.
A major downside of this methodological approach is that
experiments that make use of the fully competent virus must
be carried out at biosafety containment level 2 or 3, depending
on local regulations (Ugolini, in press). However, pseudora-
bies virus can be used for transsynaptic tracing at lower
biosafety levels.
3. Malaria and the brain
3.1. Malaria today
Malaria is one of the most important global health problems
and, together with tuberculosis and HIV/AIDS, is one of the
three infectious diseases that exert the highest impact
worldwide in terms of morbidity, mortality, and socio-
economic consequences. Malaria is the most prevalent
parasitic disease in the world. The disease is caused by the
obligate intracellular apicomplex protozoan of the genus
Plasmodium, of which four species are human pathogenic:
Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae,
and Plasmodium ovale. The parasites are transmitted to humans
by bites of infected female mosquitoes of the genus Anopheles.
In 2008, there were an estimated 243 million cases of
malaria worldwide, with more than three billion individuals
at risk (WHO World Malaria Report 2009), and the vast
majority of cases (85%) occurring in Africa. Malaria accounted
for an estimated 863,000 deaths in 2008, 89% of which were in
Africa. The majority of the infections in Africa are caused by
P. falciparum, and the blood-stage cycle of P. falciparum causes
the most severe manifestations of the disease and accounts
for the majority of deaths (Combes et al., 2010).
Several features of malaria and the immune response to it
suggest that the parasite and the immune system interact
through numerous complex mechanisms. It is important to
consider that P. falciparum is estimated to be >100,000 years
old, i.e., as old as Homo sapiens, suggesting that the human
immune system and the parasite coevolved. Indeed, malaria
has exerted more impact than any other pathogen in shaping
the human genome (Pierce and Miller, 2009).
The infection of human erythrocytes is ultimately respon-
sible for all the clinical pathologies associated with malaria.
The overall life cycle of the parasite, whose clarification has
historically required many efforts (see Section 3.2), is now well
known, and will be here summarized because of its impor-
tance for disease pathogenesis. It is a complex cycle that
involves both the insect vector and the human host.
Motile P. falciparum sporozoites are injected into the human
blood stream by female Anopheles mosquitoes during a blood
meal and rapidly invade hepatocytes. After 10–12 days,
infected hepatocytes rupture and release thousands of
daughter merozoites back into the blood, where they invade
erythrocytes and undergo the intra-erythrocytic cycle of
asexual replication during the following 48 h.
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P. falciparum differs from other human malarial species in
that parasitized red blood cells (PRBCs) do not remain in the
circulating blood for their entire life cycle. After 24–32 h, when
young parasites mature to the trophozoite stage, PRBCs adhere
to endothelial cells in the microcirculation of various organs, a
phenomenon called sequestration. Trophozoites then mature
into schizonts, which rupture and release daughter merozoites
that invade fresh erythrocytes to perpetuate the asexual life
cycle. Some parasites inside RBCs differentiate into male and
female gametocytes. Upon ingestion by a feeding female
mosquito, they fuse, undergo complete sexual development,
and form infective sporozoites that can be introduced into the
human host at the next blood meal, thereby ensuring the
continuation of the parasite life cycle.
Due to the process of sequestration, which prevents PRBCs
from being destroyed by the spleen, PRBCs tend to disappear
from the free circulation, causing a drop in the observed
peripheral parasitemia, and localize preferentially in the deep
vascular beds of body organs (brain, lung, gut, and heart).
Sequestration is thought to be due to a specific interaction
between PRBCs and the vascular endothelium. This phenom-
enon, called cytoadherence, is mediated by parasite-encoded
adhesion ligands (the family of proteins belonging to
P. falciparum erythrocyte membrane protein-1, PfEMP-1), which
allow the parasite the evade host immune responses and
mediate adhesion to host receptors, including the intercellular
adhesion molecule (ICAM)-1 (Medana and Turner, 2006).
been found inhabiting human blood cells (Cox, 2010). Laveran,
however, persevered, and visited also the group of malarial-
ogists at the University of Rome: Ettore Marchiafava (1847–
1935), Angelo Celli (1857–1914), Amico Bignami (1862–1929),
and Giovanni Battista Grassi (1854–1925). During his medical
studies, Grassi had been the first intern student in Golgi's
laboratory. In 1885 Marchiafava and Celli, convinced that
malaria was caused by a parasite, made a fundamental
contribution to the definition of the protozoan that they called
Plasmodium.
The laboratory of the Roman school of malariology was
also visited by Golgi in 1885. The contributions of Camillo
Golgi to malaria, between 1885 and 1892, have been
repeatedly reviewed (Santamaria, 1994; Mhlanga et al.,
1997; Tognotti, 2007; Muscatello, 2007; Mazzarello, 2010)
and are here briefly summarized. Golgi studied the repro-
duction of Plasmodium in the human blood. He differentiated
between tertian (48 hour periodicity) and quartan (72 hour
periodicity) malaria. Using thin smears of fresh blood, Golgi
discovered the asexual development and reproduction of
the parasites responsible for quartan (P. malariae) and tertian
(P. vivax) fevers. Furthermore, Golgi showed and elucidated
the temporal coincidence between the recurrent fever bouts
and the rupture of the erythrocytes and release of merozoites
in the blood stream (Fig. 5). This fundamental observation

3.2. Camillo Golgi and malaria

The history of malaria, like that of rabies, extends into

antiquity. The bouts of fever of malaria are recorded from
every civilization (Cox, 2002), including writings in Mesopo-
tamia, in which symptoms reminiscent of cerebral malaria
are also mentioned (Fales, 2010). The name of the disease
(from the Latin mala aria, bad air) reflects the old miasma
theory of the origin of the disease potentially produced by
noxious vapors emitted by stagnant water. As for other
infectious diseases, the understanding of malaria did not
begin until the late 1870s, with the introduction of the germ
theory of infection by Louis Pasteur and Robert Koch (1843–
1910) and the birth of microbiology. The search for the cause
of microbes responsible for infectious diseases, including the
microorganism responsible for malaria and the route of
infection, intensified then in medical laboratories around the
world (Cox, 2010; Mazzarello, 2010). Malaria was prevalent in
many areas of Italy, and the Italian Corrado Tommaso-
Crudeli and the German Theodor Albrecht Edwin Klebs
claimed to have isolated a bacterium (Bacillus malariae) from
the waters of the Pontine Marshes, near Rome, stimulating a
heated debate.
The milestone in the history of malaria is represented by
the observation of Charles Louis Alphonse Laveran (1845–
1922), a French army surgeon working at the Military Hospital
of Constantine, in Algeria, who observed the parasites in the Fig. 5 – Drawing of Camillo Golgi showing the relationships of
blood of patients and was immediately convinced that they fever in quartan (top) and tertian (bottom) malaria with the
were the etiological agents of the disease. He communicated parasite cycle in erythrocytes. The Italian text emphasizes
his findings in December 1880 to the French Academy of the “demonstration of the coincidence of the fever bout with
Medical Sciences (Laveran, 1881). Laveran's announcement the multiplication of parasites”. Courtesy of the Museum for
was received with skepticism: no protozoan had previously the History of the University of Pavia.
164 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3

was named by his mentor, Giulio Bizzozero, “Golgi's law”.
Golgi also identified the phagocytic phenomena occurring
in the spleen as an active response of the organism to limit
the infection. Importantly, Golgi proposed that quinine had
to be administered during the process of segmentation of
the parasite and therefore a few hours before the onset of
fever.
Ronald Ross (1857–1932), a Surgeon-Major in the British
Indian Medical Service, under the stimulus and suggestions of
Patrick Manson (1844–1922), discovered in 1897 that culicine
mosquitoes transmitted the avian malaria parasite. In 1898,
Grassi demonstrated that the Anopheles mosquito is the vector
of human malaria and described the Plasmodium cycle in this
primary host.
Ross received the Nobel Prize in Physiology or Medicine in
1902, and Laveran in 1907. Grassi was repeatedly nominated
for the Nobel Prize but did not receive it. The long history of
discoveries of malaria has a number of controversies, includ-
ing a correspondence between Grassi and Golgi (Mazzarello,
2010), and a long correspondence between Manson and Ross
(Cox, 2010). There is no doubt, however, that Golgi (who, as it is
well known, received the Nobel Prize in 1906 with Cajal for his
studies on the nervous system, and not for his studies on
malaria) made fundamental contributions to malaria at a time

Fig. 6 – First illustration of cerebral malaria: drawing from the pioneering study by Marchiafava and Celli (1887), showing
Plasmodia (as stated in the original figure legend) within “small blood vessels” of the cerebral cortex in cases of malaria, with
images of parasite replication, pigment, and alterations of erythrocytes.
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
when knowledge of the relationships between the malaria
parasite and the infected patient were very “nebulous” and
“intricate” (Bertarelli, 1950).
Sequestration was first recognized and illustrated by
Marchiafava and Celli (1887) and Marchiafava and Bignami
(1892). The monograph by Marchiafava and Bignami (1892) on
the “summer-autumnal fever” (the Italian name for malig-
nant, i.e., P. falciparum, malaria) was translated by the New
Sydenham Society and provided the foreign scientific com-
munity with a synopsis of the Italian work.
Laveran (1893) realized the involvement of the brain in
malignant malaria and Marchiafava and Bignami (1892)
clearly described clinical symptoms of cerebral malaria. The
article by Marchiafava and Celli (1887) provided the first
illustration of sequestration in cerebral blood vessels (Fig. 6).
3.3. Cerebral malaria
Cerebral malaria, which is part of the heterogeneous syn-
drome called severe malaria, is a life-threatening neurological
syndrome caused by P. falciparum infection of the CNS. The
disease occurs predominantly in patients with little or no
background immunity, including children growing up in
endemic areas of Africa, or adults who have not acquired
immunity to malarial infection or have lost their immunity to
the disease (Gitau and Newton, 2005).
Cerebral malaria is clinically defined as a deep level of
unconsciouness (unarousable coma), often associated with
seizures (especially in children), in the presence of P. falcip-
arum asexual parasitemia and in the absence of other
factors that could cause unconsciousness, such as hypogly-
cemia and exclusion of other encephalopathies, especially
microbial ones such as bacterial meningitis (Newton, 2000;
Medana and Turner, 2006).
Cerebral malaria is potentially reversible. Most African
children with cerebral malaria survive with appropriate
treatment, regaining consciousness within 2 or 3 days of
starting treatment. Cerebral malaria is, however, fatal in 15–
30% of the cases. Among the survivors, more than 10% of the
children affected by cerebral malaria have neurological
sequelae, which can be severe and fatal within a few months
(spastic tetraparesis, vegetative states), or are represented by
more subtle deficits such as cognitive difficulties, language,
and behavioral problems. Long-term cognitive impairments
(primarily attention deficits) have been reported in 1 of 4 child
survivors of cerebral malaria (John et al., 2008). The causes of
the sequelae are largely unknown and are likely to be
multifactorial (Newton, 2000). Adults affected by severe
malaria may die from non-cerebral (pulmonary or renal)
rather than from neurological complications. Less than 5% of
adults surviving cerebral malaria have neurological deficits
and these are usually minor, such as cranial nerve palsy (Gitau
and Newton, 2005).
A retrospective study of levels in the cerebrospinal fluid of
2 markers of brain parenchymal damage in Kenyan children
with several malaria (Medana et al., 2007) gave interesting
results. Levels of the microtubule-associated protein tau as
marker for degenerated axons were found to be inversely
correlated with the level of consciousness and other para-
meters of severity of the infection, pointing to an association
165
of axonal injury with the severity of the clinical picture. High
levels of S-100B as marker of astrocytes were associated with
an increased risk of repeated seizures, supporting an implica-
tion of astrocytes with seizure activity, but not with other
clinical parameters.
3.3.1. The puzzle of the pathology and pathogenesis of
cerebral malaria
Comprehensive treatises of the neuropathology of cerebral
malaria have been published (e.g. Turner, 1997; Newton,
2000; Haldar et al., 2007). Since, as mentioned above, the
Plasmodium develops within RBCs and remains inside the
vascular space, the parasite does not enter the brain
parenchyma. The brain is especially affected by the phe-
nomenon of sequestration (Fig. 2C). The central pathological
feature of cerebral malaria is thus represented by the
engorgement of the cerebral microvasculature (cerebral
capillaries and venules) with PRBCs and non-parasitized
RBCs. Distended venules containing PRBCs are prominent in
the brain grey matter. However, the variability of histopath-
ological features is a puzzling aspect of cerebral malaria:
cerebral sequestration has also been found in victims of non-
neurological complications of P. falciparum malaria, and
brains from cases affected by cerebral malaria may lack
histological evidence of sequestration.
Rosetting (i.e., the adherence of non-parasitized RBCs to
PRBCs) and agglutination (i.e., the adherence of PRBCs to
PRBCs) (Fig. 2C) have also been implicated in the pathogenesis
of cerebral malaria. Furthermore, both PRBCs and non-
parasitized RBCs become less deformable upon parasite
growth within the RBCs, which may also contribute to
impairment of microcirculatory flow.
In human victims, hemorrhages are found in the brain
(Fig. 2C), as punctiform or petechial hemorrhages and ring
hemorrhages. In petechial hemorrhages, normal RBCs sur-
round a ruptured cerebral vessel. In the brain, petechiae are
preferentially found in the white matter; they can also be
found, although less frequent, in cases of malaria who do not
meet the clinical criteria of cerebral malaria, and can also be
seen in other organs. Ring hemorrhages are instead a
neuropathological feature unique to malaria (Turner, 1997)
and consist of a series of concentric rings surrounding a
central necrosed blood vessel. The outermost ring contains a
mixture of PRBCs, free pigment and host monocytes, and the
inner layer contains non-parasitized RBCs and gliosis sur-
rounding the vessel. Petechial hemorrhages may be the result
of vessel rupture in areas of no sequestration, where parasites
and their products have not elicited a host reaction, in contrast
with Dürck granuloma, which is another feature peculiar to the
brain in malaria (Turner, 1997). Originally attributed to glial
cell proliferation associated with hemorrhagic necrotic areas,
the granuloma could represent a glial and immune reaction in
the parenchyma due to leakage of exudate, cell, and parasites
caused by endothelial cell damage, or could represent a
temporal evolution of ring hemorrhages.
Cerebral malaria is associated with a Th1 immune
response, in which the proinflammatory cytokines IFN-γ and
TNF-α, as well as CD4+ and CD8+ T cells and natural killer cells
play major roles (Hunt and Grau, 2003; Hunt et al., 2006).
However, transendothelial migration of lymphocytes and
166 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
macrophages does not occur during cerebral malaria, in
which, therefore, brain pathology is mediated essentially by
intravascular inflammatory events, at variance with other
brain infections (Bruzzone et al., 2009). Induction of the
transcripts encoding the proinflammatory cytokines TNF-α
and IL-1β has been documented in the brain of cases of
cerebral malaria but was not found to correlate with seques-
tration (Medana and Turner, 2006).
Hypertrophy of perivascular and parenchymal microglia
and astrocytes (Fig. 2C) has been observed in the brain of
victims of cerebral malaria and has also been documented in
cases of non-cerebral malaria (Medana et al., 2001; Medana
and Turner, 2006). Considerable variability, however, occurs in
the response of microglia and astrocytes in fatal cases of
severe malaria without neurological complications and in
cases of fatal cerebral malaria.
The problem of murine models of cerebral malaria adds to
the complexity of pathogenetic studies. Because P. falciparum
does not infect murine hosts, different Plasmodium species
are used to infect laboratory mice. The most widely used model
is represented by infections of C57BL/6 or CBA mice with
Plasmodium berghei ANKA strain. The mice die in the second
week of infection from encephalopathy associated with
seizures. This murine infection has a number of differences
with the human disease (White et al., 2009). In particular,
erythrocytes sequester mainly in the lungs and adipose tissue
rather than in the brain, and leukocytes and platelets markedly
accumulate in the brain venules and capillaries.
Although differences between the human and murine
pathology require a critical consideration, observations in the
murine model of fatal cerebral malaria point to an involvement
of glial activation in the disease (Medana and Turner, 2006;
Szklarczyk et al., 2007). For example, an experimental increase
in BBB permeability in this murine model was found to elicit
thickening of microglial processes and redistribution of
microglia toward the vasculature (Medana et al., 2000).
Concerning the mechanisms which could activate glial
cells, adhesive interactions between PRBCs and cerebral
endothelia are likely to include localized hypoxia and endo-
thelial cell activation with increased release of proinflamma-
tory molecules that can act on perivascular and parenchymal
glia (Szklarczyk et al., 2007). Loss of cell junction proteins
seems to cause leakage at the BBB, with protein and liquid
diffusion in the brain tissue (Rasti et al., 2004). The changes in
the CNS vasculature could influence astrocytes and microglia,
inducing the release of inflammatory mediators during the
disease process, which could be reinforced by cytokines
derived from monocytes adhering to the vascular endotheli-
um. Cytotoxic factors produced by leukocytes interacting with
the vascular endothelium could damage astrocytes adhering
to blood vessel walls (Fig. 2C).
4. Human African trypanosomiasis: sleep and
wake in flame
4.1. HAT today
The disease HAT, also known as sleeping sickness, is caused
by a subspecies of the extracellular single-celled parasites
Trypanosoma brucei (T.b.), which belong to the Salivarian group of
the genus Trypanosoma. Tsetse flies (genus Glossina) are the
vectors of the parasites. Foci of the disease occur in suitable
habitats for this vector in sub-Saharan Africa, mainly poor and
remote rural regions. The parasite subspecies T.b. gambiense
causes the vast majority of human infections, with a chronic
form of disease lasting several months or years, in West and
Central Africa. Humans are the primary host and main
reservoir. The subspecies T.b. rhodesiense causes a more acute
form of the disease in East and Southern Africa, for which wild
game animals and cattle are the main reservoir. Except for the
temporal evolution of the disease, both forms of HAT exhibit
similar clinical and pathological features.
A third subspecies of the parasite, T.b. brucei, produces a
relatively mild form of disease in native game animals but a
severe condition in most domesticated animals. T.b. brucei is
non-infective for humans, who have a serum protein that
inhibits this parasite, but produces a lethal disease in rodents,
and is therefore used in experimental studies.
The 3 subspecies of T.b. are morphologically and to the
main serologically indistinguishable. They are covered on
their surface by a single species of variant surface glycopro-
teins (VSGs) and express a new species of VSG on their
surface after each immune attack. By this mechanism, the
parasites evade the host humoral immune response and can
avoid to be eliminated in mammals. The VSG genes occupy
10% of the trypanosome genome and the mechanisms for
the process of VSG switching are well known. The sequence
of the 26-megabase genome of T.b. has been completed in
2005.
African trypanosomes multiply in the host blood and are
taken up by tsetse flies when they feed. Within the tsetse fly,
there is a phase of multiplication and development of the
parasites; this results in the formation of infective parasites in
the salivary gland of the fly, which are injected into a new host
when the fly feeds.
HAT, which is invariably fatal if left untreated, causes a
considerable burden in endemic countries, with 1.54 million
DALYs lost in 2002 (Fèvre et al., 2008). As mentioned further
(see Section 4.2), the disease occurs in outbreaks, which
reflect shortcomings in surveillance and treatment due to
political instability and civil unrest, increased migration of
human populations due to streams of refugees, as well as
disruption of tsetse fly control programs. The number of cases
has recently decreased, and 50,000–70,000 cases are currently
reported, but this number should be regarded with caution
due to under-reporting (Brun et al., 2010). The decrease in the
number of reported cases has raised the concern that it could
lead to a reduction in the efforts for case detection, which has
led in the past to disease resurgence (Fèvre et al., 2008;
Simarro et al., 2008; see also Section 4.2). A recent character-
ization of the association between HAT incidence and
conflict-related processes in African countries affected by
HAT over the past 30 years (Berrang-Ford and Breau, 2010) has
confirmed the predominant coincidence of clusters of HAT
incidence with periods of conflict or socio-political instability.
It is also worrying to note that the results of this study have
indicated a relatively long lag period, of approximately
10 years, between the start of conflict events and the peak
of HAT incidence, indicating that surveillance should be
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
actively pursued for many years to achieve control of the
disease.
The clinical picture of HAT is polymorphous and the
disease evolves in two stages (Kennedy, 2008). The parasites
multiply in the subcutaneous tissue at the site of the infesting
bite, and then gradually spread to the lymphatics and blood
stream, where they continue extracellular replication. The
signs of this first systemic, hemolymphatic stage are not
characteristic, and include fever, headache, pruritus. The
disease then progresses to the second, meningoenceph-
alitic stage when the parasites cross the BBB and invade
the brain parenchyma. A complex neuropsychiatric syn-
drome develops, with a constellation of motor, sensory, and
mental disturbances. A characteristic symptom of HAT is
represented, however, by disturbances of sleep and wake,
represented by diurnal somnolence and nocturnal insomnia,
with disturbances of sleep and wake circadian alternation as
well as of sleep structure (Lundkvist et al., 2004; Kristensson
et al., 2010).
Drugs effective in curing HAT early in the infection are
available, but they do not, or poorly, cross the BBB. The arsenic
compound melarsoprol is widely used to cure second stage
HAT, but this drug is highly toxic and can cause a post-
treatment acute reactive encephalopathy which leads to
death 5–10% of the patients. As also highlighted recently
(Bruzzone et al., 2009; Kristensson et al., 2010), diagnostic tools
to identify early HAT infection, and non-toxic drugs to cure the
brain infection are urgently needed.
4.2. Historical note
Several historical documents indicate that HAT is, as rabies
and malaria, a very old disease, present in the African
continent since ancient times. The more recent and better
documented history of the disease dates, however, to the
colonial times (Cox, 2002; Kennedy, 2007; Sterverding, 2008).
Sizeable epidemics in humans were identified at the turn
of the 20th century. It has been estimated that half a
million people died of sleeping sickness along the Congo
river at the time of its exploration between 1896 and 1908.
The most renowned outbreak of the disease occurred on
the northern shore of Lake Victoria between 1898 and 1908.
This epidemic may have killed two-thirds of the population
in Uganda and the hunt for its causative agent was,
therefore, intensified.
Only two protagonists of the variegated history of sleeping
sickness are here mentioned for brevity, and both of them are
connected to the discovery of the etiological agent and its
vector. One of them is the Italian bacteriologist Aldo
Castellani (1874–1971), who was to become a famous tropic-
alist and clinician (Bentivoglio et al., 1994). Castellani was a
member of the First Sleeping Sickness Commission sent in
1902 by the Royal Society to Uganda to investigate the
devastating epidemic of the disease that was decimating
the local population. He observed the trypanosomes in the
cerebrospinal fluid of sleeping sickness patients, and com-
municated his observations to the Royal Society (Castellani,
1903) (Fig. 7). His findings were, however, received with some
skepticism. The Royal Society sent then to Uganda David
Bruce (1855–1931) as member of the Second Sleeping Sickness
167
Commission. Bruce confirmed Castellani's discovery, demon-
strated that the disease was transmitted by tsetse flies,
and the parasite was named after him Trypanosoma brucei. As
in the history of malaria, and in the history of other
discoveries, the priority of the above discoveries for sleeping
sickness has been controversial (Boyd, 1973; Kennedy, 2007),
but both Castellani and Bruce certainly deserve recognition.
Castellani insisted on his priority also concerning the name of
the parasite and the disease, that he denominated Castella-
nella (Fig. 7) and castellanosis, respectively (Castellani and
Jacono, 1937).
Following the widespread and severe outbreaks during the
late 1800s and early 1900s, HAT was virtually eliminated in the
1960s during the colonial period, largely due to mobilization of
considerable resources for the control of the disease. Reemer-
gence of HAT in the 1970s corresponded with a period of post-
independence civil unrest and instability in many African
countries, which was associated with rapid decreases in
funding for HAT prevention and control (Simarro et al.,
2008). As also mentioned previously, the number of cases
has decreased in the last few years.
4.3. Neuropathology of African trypanosomiasis
Meningoencephalitis with diffuse inflammatory changes,
which prevail in the periventricular areas and white matter
of the cerebral hemispheres, has been observed in human
victims of HAT. Inflammatory cell infiltrates in the lepto-
meninges and around blood vessels in the brain parenchy-
ma have been reported in T.b.-infected human cases since
early pathological studies (Mott, 1907; Spielmeyer, 1908) and
in more recent post-mortem investigations (Brown and
Voge, 1982). Perivascular cuffs are mainly formed by
lymphocytes and plasma cells. Morular (mulberry-like) cells
(Mott's cells), which contain immunoglobulins of the IgM
type are associated with clusters of inflammatory cells in
the T.b.-infected brains, but are also observed as isolated
cells in the parenchyma. They also occur in other chronic
inflammatory brain diseases. Reactive gliosis has also been
observed (Spielmeyer, 1908; Brown and Voge, 1982), with
diffuse proliferation and hypertrophy of astrocytes, and
activation of microglial cells with the formation of microglial
nodules (Chinelli and Scaravilli, 1997). Although some
neuronal damage can be observed (Brown and Voge, 1982),
neuronal sparing at the histopathological examination of the
infected human brains is in contrast with the severe and
progressive neurological picture of the disease (Chinelli and
Scaravilli, 1997; Kristensson et al., 2010). Histopathological
features of a non-specific meningoencephalitis have also
been described in the post-treatment arsenical encephalop-
athy (Adams et al., 1986).
4.4. Neuroinflammatory signaling and African
trypanosome infection
High levels of inflammatory cytokines, and in particular IFN-
γ, TNF-α, and IL-1β, as well as of anti-inflammatory cytokines
such as IL-10, have been documented in the brain of T.b.
brucei-infected rodents, and in the cerebrospinal fluid and
blood of HAT patients (Kennedy, 2009; Kristensson et al.,
168 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3

Fig. 7 – Trypanosoma brucei at the time of the discovery of parasites in the cerebrospinal fluid of patients affected by human
African trypanosomiasis (HAT) and today. Left: Drawings of the different appearance of the parasite Trypanosoma brucei
gambiense observed in Uganda by Aldo Castellani in the cerebrospinal fluid of patients affected by the West African form of HAT,
which illustrated Castellani's report to the Royal Society (Reports of the Royal Society Sleeping Sickness Commission, No. 2,
November 1903). The image is reproduced from Castellani and Jacono (1937) and the image legend states “Morphological
variations of Castellanella gambiensis… 1–5. Typical adult forms. 6–8. Atypical adult forms. 9–11. Forms in various stages of
longitudinal division. 12–14. Different types of forms of development or fusion”. As stated in the text, Aldo Castellani
denominated the parasite after himself, whereas the parasite had actually been denominated Trypanosoma brucei after David
Bruce. Right: Confocal microscopy images of Trypanosoma brucei brucei in the brain parenchyma of the rat brain after
experimental infection, as visualized by double immunofluorescence with antibodies that recognize the parasite (green), and
glucose transporter-1 (Glut-1) as marker of walls of capillaries.

2010). The disease is, therefore, dominated by inflammatory
mediators and their delicate balance. Data and open ques-
tions in disease pathogenesis have been recently reviewed
(Kristensson et al., 2010). Only main findings related to glia
and infiltrating T cells are mentioned here.
Demyelination and modest, if any, signs of degeneration of
neuronal cell bodies have been found also in the brain of T.b.
brucei-infected rats and mice, even in chronic forms of
experimental infection (Keita et al., 1997; Quan et al., 1999).
Degenerating fibers have been revealed by silver staining at
certain brain sites in T.b. brucei-infected rats, indicating that
axonal degeneration may occur in this model (Quan et al.,
1999). Experimental manipulations have indicated that TNF-α,
together with IL-1β, could be a main mediator of these
neurodegenerative events (Quan et al., 2003).
It was observed in experimental paradigms of infection
that African trypanosomes reside in the choroid plexus and
circumventricular organs located outside the BBB before
invading the brain. The parasites then traverse the BBB
(Fig. 7) without a generalized loss of tight junction proteins
(Mulenga et al., 2001). Lymphocyte-derived IFN-γ facilitates
the penetration of the parasites across cerebral blood vessels
(Masocha et al., 2004). This inflammatory cytokine, which is
involved in stimulating the immunological control of the
infection, also has, paradoxically, an effect in promoting
neuroinvasion of the parasite. Furthermore, the composition
of the basement membranes of cerebral blood vessels has a
key role in determining the sites of T.b. penetration into the
brain (Masocha et al., 2004, 2007). Host genes encoding
inflammatory molecules are involved in determining T.b.
brucei invasion of the brain parenchyma, which is instead
unrelated to parasitemia or antibody titres (Masocha et al.,
2008). Lymphocytes are required for migration of the parasite
into the brain, indicating that African trypanosomes utilize
mechanisms similar to those of leukocytes to penetrate the
BBB and infiltrate the brain parenchyma, or that leukocytes
could alter the structural integrity of the basement mem-
branes paving the way to parasite entry (Masocha et al., 2007;
Kristensson et al., 2010). The multi-step process of African
trypanosome and T cell entry into the brain (Fig. 2D) is
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
crucial in the severe morbidity of the disease (Kristensson
et al., 2010).
Activation of astrocytes has been detected in murine
models of T.b. brucei infection (Hunter et al., 1992a; Keita
et al, 1997) (Fig. 2D) and of post-treatment encephalopathy
(Hunter et al, 1992b). The timing of astrocytic activation and its
concomitance with the production of inflammatory cytokines
has suggested that astrocytes could represent a source of
these molecules in the brain during the infection (Hunter et al.,
1992a). Both astrocytes and microglia, as well as perivascular
monocytes, express the inhibitory factor KBα (IKBα) at ad-
vanced stages of T.b. brucei infection in rats (Quan et al., 1999).
The IKBα is an immediate early gene responsive to stimulation
of inflammatory cytokines, and the finding, therefore, further
indicated that astrocytes and microglia contribute to the
regulation of cytokine synthesis.
Investigation of microglial cells in the model of T.b.
brucei chronic infection in rats (Chianella et al., 1999) has
revealed hypertrophic microglial elements (Fig. 2D) with a
prevalence in subpial regions of the brain and in other
brain regions, and with a predominance in hypothalamic
periventricular regions. Microglia activation was not associ-
ated with histologically detectable neuronal damage. Inter-
estingly, the onset of microglia activation paralleled the
onset of sleep anomalies documented by electroencephalo-
graphic recording of the animals, and became more marked
with the progression of the disease, revealed also by
worsening of sleep alterations. Several lines of evidence
derived from recent investigations concur in pointing to a
key role of inflammatory signaling, mediated by molecules
released by glia and T cells during the infection, in targeting
the function of neurons implicated in the regulation of
sleep and wakefulness and endogenous biological rhythms
(Kristensson et al., 2010).
Many questions remain open in the crosstalk between
neurons and non-neuronal resident and infiltrating cells
during African trypanosome infection. However, early local-
ization of the parasites to basal meninges and circumven-
tricular organs may cause the release of molecules that affect
neuronal subsets. Glial activation in regions of the brain
parenchyma could be involved in this interplay also during
the progression of the disease. Such molecules include not
only inflammatory and anti-inflammatory cytokines and
chemokines released by the host during the immune
response to the parasites, but also molecules, such as
prostaglandin D2, which can be released by the parasites
(Kristensson et al., 2010).
In the brain infected by T.b. brucei, minocycline treatment
inhibits infection-induced upregulation of transcripts
expressed by activated microglia (Masocha, 2010), indicating
that this drug can modulate microglia activation during the
infection. Interesting data have also been recently obtained in
the study of chemokines released during the disease, and
studies in humans and experimental models of African
trypanosomiasis have pointed to CXCL10 as potential bio-
marker of the encephalitic stage of the disease (Amin et al.,
2009; Hainard et al., 2009). In the present context, it is
interesting to note that in the murine model of the infection
CXCL10 was found to be predominantly expressed in astro-
cytes (Amin et al., 2009).
169
5. Concluding remarks
The inflammatory response is a cardinal defense of the
organism against injury, planned to remove or sequester the
source of disturbance, to allow the host to cope with the
abnormal conditions, to restore tissue functionality and
homeostasis (Medzhitov, 2008). Although neuroinflammation
has features different from those of peripheral inflammation
due to the immune specialization of the CNS and involves
different cell types, neuroinflammation is, as peripheral
inflammation, primarily a defense response which builds up
to protect the CNS. During chronic inflammation, however, the
abnormal conditions are sustained and can become maladap-
tive for the organism (Medzhitov, 2008). For the brain
microenvironment, in which the immune response is normally
downregulated due to immune specialization, this is especially
crucial.
In rabies, neural functioning seems to be exploited by the
causative virus to its own advantage, and the virus fights to
suppress the dialogue between neurons and non-neuronal
cells. The intracellular Plasmodium and leukocytes do not
infiltrate the brain parenchyma in cerebral malaria, and the
pathogenetic role of astrocytes and microglia in this disease is
still unclear. The extracellular African trypanosome and
leukocytes invade the brain parenchyma in the encephalitic
stage of African trypanosomiasis, but the role of astrocytes,
microglia, and infiltrating T cells in T.b. infection remains to
be fully clarified. This role is probably fundamental not only
for the morbidity of HAT, but also in the functional targeting
of neuronal subsets and in the release of distinct inflamma-
tory mediators. In all these diseases, the functioning of the
brain parenchyma is affected by an interplay between
neurons and non-neuronal cells, which seems to largely
spare neuronal structure while deeply affecting neuronal
functions.
As mentioned initially (see Section 1.2), chronic neuroin-
flammation is currently viewed as closely associated to
chronic neurodegenerative diseases. CNS infections show,
however, that the inter-relationships between neuroinflam-
mation and neuronal cell death or survival are complex and
multifaceted. Pathogenetic mechanisms of neurodegenera-
tion should help in answering the numerous open questions
on the pathogenesis of neuronal damage in non-neurode-
generative brain infections which currently do not seem to
raise much interest in the neuroscience community. Knowl-
edge on the “non-cell autonomous” mechanisms operant in
brain infections could feed into knowledge on intercellular
dialogue in neurodegeneration. Highly multidisciplinary and
integrated approaches should converge, in the neuroscience
community, in the fight against these diseases.
Acknowledgments
The preparation of this manuscript has been in part supported
by NIH/Fogarty grant number 1R21NS064888-01A1. The
authors are very grateful to Javier DeFelipe, Livia Iannucci,
Monique Lafon, and Marco Piccolino for their invaluable help
with historical bibliographic material on rabies and malaria.
170 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
Thanks are also due to Maria Palomba for her help in the
confocal microscopy images of Trypanosoma brucei brucei in the
brain parenchyma.
REFERENCES
Abbott, N.J., Rönnbäck, L., Hansson, E., 2006. Astrocyte–endothelial
interactions at the blood–brain barrier. Nat. Rev. Neurosci. 7,
41–53.
Adams, J.H., Doua, A., Dago-Akribi, A., Boa, Y., Haller, L., 1986.
Human African trypanosomiasis (T.b. gambiense): a study of 16
fatal cases of sleeping sickness with some observations in
acute reactive arsenical encephalopathy. Neuropathol. Appl.
Neurobiol. 12, 81–94.
Amin, D.N., Rottenberg, M.E., Thomsen, A.R., Mumba, D., Fenger,
C., Kristensson, K., Büscher, P., Finsen, B., Masocha, W., 2009.
Expression and role of CXCL10 during the encephalitic stage of
experimental and clinical African trypanosomiasis. J. Infect.
Dis. 200, 1556–1565.
Arck, P.C., Gilhar, A., Bienenstock, J., Paus, R., 2008. The alchemy
of immune privilege explored from a neuroimmunological
perspective. Curr. Opin. Pharmacol. 8, 480–489.
Babes, V., 1892. Sur certain charactères des lesions histologiques
de la rage. Ann. Inst. Pasteur 6, 209–223.
Bentivoglio, M., 1999. The discovery of axonal transport. Brain Res.
Bull. 56, 383–384.
Bentivoglio, M., 2003. Intraneuronal inclusion bodies: from Negri
bodies to proteasomal dysfunction. Rend. Fis. Acc. Lincei 14,
263–279.
Bentivoglio, M., Kristensson, K., 2004. Thalamus in flame:
targeting of infectious agents to thalamic nuclei. Thal. Rel.
Syst. 2, 297–314.
Bentivoglio, M., Grassi-Zucconi, G., Kristensson, K., 1994. From
trypanosomes to the nervous system, from molecule to
behavior: a survey, on the occasion of the 90th anniversary
of Castellani's discovery of the parasites in sleeping sickness.
Ital. J. Neurol. Sci. 15, 77–89.
Berrang-Ford, L., Breau, L.J., 2010. Conflict and human
trypanosomiasis. Soc. Sci. Med. (Epub ahead of print June 23).
Bertarelli, E., 1904a. Sopra le vie per le quali il virus rabido
arriva alle ghiandole salivari del cane. Arch. Sci. Med. 28,
179–191.
Bertarelli, E., 1904b. Ueber die Wege, auf denen das Wutvirus zu
den Speicheldrüsen des Hundes gelangt. Centralbl. Bakt.
Parasit. Infektionskrankh. 37, 213–221.
Bertarelli, E., 1906a. Sulla trasmissione della sifilide al coniglio. Riv.
Ig. San. Pubbl. 17, 1–7.
Bertarelli, E., 1906b. Ueber die Transmission der Syphilis auf das
Kaninchen. Centralbl. Bakt. Parasit. Infektionkrankh. 41,
320–326.
Bertarelli, E., 1950. Camillo Golgi ed il suo tempo nel venticinquesimo
anniversario della sua morte. Istituto Sieroterapico Milanese S.
Belfanti, Milan.
Bertarelli, E., Volpino, G., 1903. Morphologische und biologische
Beobachtungen über einen Fall von Wutkrankheit beim
Menschen, mit besonderer Rücksicht auf die Gegenwart und
Verteilung der Negrischen örpechen in Zentralnervensystem.
Centralbl. Bakt. Parasit. Infektionkrankh. 35, 221–223.
Block, M.L., Zecca, L., Hong, J.-S., 2007. Microglia-mediated
neurotoxicity: uncovering the molecular mechanisms. Nat.
Rev. Neurosci. 8, 57–69.
Boillée, S., Vande Valde, C., Cleveland, D.W., 2006. ALS: a disease of
motor neurons and their nonneuronal neighbors. Neuron 52,
39–59.
Boyd, J., 1973. Sleeping sickness. The Castellani–Bruce controversy.
Notes Rec. R. Soc. Lond. 28, 93–100.
Brown, W.J., Voge, M., 1982. Neuropathology of Parasitic
Infections. Oxford University Press, Oxford.
Brujin, L.I., Becher, M.W., Lee, M.K., Anderson, K.L., Jenkins, N.A.,
Copeland, N.G., Sisodia, S.S., Rothstein, J.D., Borchelt, J.D., Price,
D.L., Cleveland, D.W., 1997. ALS-linked SOD1 mutant G85R
mediates damage to astrocytes and promotes rapidly
progressive SOD1-containing inclusions. Neuron 18, 327–338.
Brun, R., Blum, J., Chappuis, F., Burri, C., 2010. Human African
trypanosomiasis. Lancet 375, 148–159.
Bruzzone, R., Dubois-Dalcq, M., Grau, G.E., Griffin, D.E., Kristensson,
K., 2009. Infectious diseases of the nervous system and their
impact in developing countries. PLoS Pathog. 5 (2), e100199.
doi:10.1371/journal.ppat.1000199 .
Buchanan, R.L., Benzer, S., 1993. Defective glia in the Drosophila
brain degeneration mutant drop-dead. Neuron 10, 839–850.
Cajal, S.R., 1996. Recollections of My Life. Translated by E.H. Craigie
and J. Cano. MIT Press, Cambridge, MA.
Cajal, S.R., García, D. Dalmacio, 1904. Las lesiones del retículo de
las células nerviosas en la rabia. Trab. Lab. Invest. 3, 213–266.
Capello, K., Mulatti, P., Comin, A., Gagliazzo, L., Guberti, V.,
Citterio, C., De Benedictis, P., Lorenzetto, M., Costanzi, C.,
Vio, P., Zambotto, P., Ferri, G., Mutinelli, F., Bonfanti, L.,
Marangon, S., 2010. Impact of emergency oral rabies
vaccination of foxes in northeastern Italy, 28 December
2009–20 January 2010: preliminary evaluation. Euro Surveill.
15 (28), pii=19617.
Carson, M.J., Thrash, J.C., Walter, B., 2006. The cellular response in
neuroinflammation: the role of leukocytes, microglia and
astrocytes in neuronal death and survival. Clin. Neurosci. Res.
6, 237–245.
Castellani, A., 1903. On the discovery of a species of Trypanosoma in
the cerebrospinal fluid of cases of sleeping sickness. Proc. R.
Soc. Lond. 71, 501–508.
Castellani, A., Jacono, I., 1937. Manuale di Clinica Tropicale.
Rosenber & Sellier, Turin.
Chianella, S., Semprevivo, M., Peng, Z.-C., Zaccheo, D.,
Bentivoglio, M., Grassi-Zucconi, G., 1999. Microglia activation
in a model of sleep disorder: an immunohistochemical study
in the rat brain during Trypanosoma brucei infection. Brain Res.
832, 54–62.
Chinelli, F., Scaravilli, F., 1997. Trypanosomiasis. Brain Pathol. 7,
599–611.
Clement, A.M., Nguyen, M.D., Roberts, E.A., Garcia, M.L., Boillée, S.,
Rule, M., McMahon, A.P., Doucette, W., Siwek, D., Ferrante, R.J.,
Brown, R.H., Julien, J.P., Goldstein, L.S., Cleveland, D.W., 2003.
Wild-type nonneuronal cells extend survival of SOD1 mutant
motor neurons in ALS mice. Science 302, 113–117.
Combes, V., El-Assaad, Faille, D., Jambou, R., Hunt, N.H., Grau, G.E.
R., 2010. Microvesiculation and cell interactions at the
brain–endothelial interface in cerebral malaria pathogenesis.
Progr. Neurobiol. 91, 140–151.
Cox, F.E.G., 2002. History of human parasitology. Clin. Microbiol.
Rev. 15, 595–612.
Cox, F.E.G., 2010. History of the discovery of the malaria
parasites and their vectors. Parasites Vectors 3. doi:10.1186/
1756-3305-3-5 .
Dietzschold, B., Schnell, M., Koprowski, H., 2005. Pathogenesis of
rabies. Curr. Top. Microbiol. Immunol. 291, 45–56.
Ehrlich, P., 1885. Das Sauerstufbudurnis des Organismus. Eine
Farbenanalytische Studie. Hirschwald, Belin, Germany.
Engelhardt, B., 2010. T cell migration into the central nervous
system during health and disease: different molecular keys
allow access to different central nervous system
compartments. Clin. Exp. Neuroimmunol. 1, 79–93.
Fales, F.M., 2010. Mesopotamia. Handbook of Clinical Neurology. : In:
Finger, S., Boller, F., Tyler, K.L. (Eds.), History of Neurology, Vol. 95
(3rd series). Elsvier, Amsterdam, pp. 15–27.
Farina, C., Aloisi, F., Meinl, E., 2007. Astrocytes are active players in
cerebral innate immunity. Trends Immunol. 28, 138–145.
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
Fèvre, E.M., Wissmann, B.V., Welburn, S.C., Lutumba, P., 2008. The
burden of human African trypanosomiasis. PLoS Negl. Trop.
Dis. 2, e333. doi:10.1371/journal.pntd.0000333 . Jackson, A.C., 2008. Rabies. Neurol. Clin. 26, 717–726.
Galea, I., Beckmann, I., Perry, V.H., 2007. What is immune privilege
(not)? Trends Immunol. 28, 12–18.
Galtier, P.V., 1879. Etudes sur la rage. Ann. Med. Vet. 28, 627–639.
Garcia-Lopez, P., Garcia-Marin, V., Freire, M., 2010. The histological
slides and drawings of Cajal. Front. Neuroanat. 4, 9.
Garden, G.A., La Spada, A.R., 2008. Molecular pathogenesis and
cellular pathology of spinocerebellar ataxia type 7
neurodegeneration. Cerebellum 7, 138–149.
Gitau, E.N., Newton, C.R.J.C., 2005. Blood–brain barrier in falciparum
malaria. Trop. Med. Int. Health 10, 285–292.
Glass, C.K., Saijo, K., Winner, B., Marchetto, M.C., Gage, F.H., 2010.
Mechanisms underlying inflammation in neurodegeneration.
Cell 140, 918–934.
Glezer, I., Simard, A.R., Rivest, S., 2007. Neuroprotective role of the
innate immune system by microglia. Neuroscience 147,
867–883.
Golgi, C., 1883. Il Sistema Nervoso Centrale. Parte I. Del Sistema
Nervoso in Generale. Generalità sul Sistema Nervoso ed
Istologia del Tessuto Nervoso. Vallardi, Milan.
Grafstein, B., 2011. Subverting the hegemony of the synapse:
complicity of neurons, astrocytes and vasculature in spread-
ing depression and pathology of the cerebral cortex. Brain Res.
Rev 66, 123–132.
Gurney, M.E., 1994. Transgenic mouse model of amyotrophic
lateral sclerosis. N. Engl. J. Med. 331, 1721–1723.
Hainard, A., Tiberti, N., Robin, X., Lejon, V., Mumba Ngoyi, D.,
Matovu, E., Enyaru, J.C., Fouda, C., Ndungu'u, J.M., Lisacek, F.,
Müller, M., Turck, N., Sanchez, J.-C., 2009. A combined CXCL10,
CXCL8 and H-FABP panel for the staging of human African
trypanosomiasis patients. PLoS Negl. Trop. Dis. 3 (6), e459.
doi:10.1371/journal.pntd.0000459 . Neurobiol. 91, 152–171.
Haldar, K., Murphy, S.C., Milner, D.A., Taylor, T.E., 2007. Malaria:
mechanisms of erythrocytic infection and pathological
correlates of severe disease. Annu. Rev. Pathol. Mech. Dis. 2,
217–249.
Hanisch, U.-K., Kettenmann, H., 2007. Microglia: active sensor and
versatile effector cells in the normal and pathologic brain. Nat.
Neurosci. 10, 1387–1394.
Hemachudha, T., Phanuphak, P., Sriwantanthana, B., Manutsahit,
S., Phanthumchinda, K., Siriprasomsup, W., Ukachoke, C.,
Rasameechan, S., Kaoroptham, S., 1988. Immunologic study of
human encephalitic and paralytic rabies. A preliminary study
of 16 patients. Am. J. Med. 84, 673–677.
Hemachudha, T., Laothamatas, J., Rupprecht, C.E., 2002. Human
rabies: a disease of complex neuropathogenetic mechanisms
and diagnostic challenge. Lancet Neurol. 1, 101–109.
Hirsch, E.C., Hunot, S., 2009. Neuroinflammation in Parkinson's
disease: a target for neuroprotection? Lancet Neurol. 8,
382–397.
Hu, R., Tang, Q., Tang, J., Fooks, A.R., 2009. Rabies in China: an
update. Vector Borne Zoonotic Dis. 9, 1–11.
Hunt, N.H., Grau, G.E., 2003. Cytokines: accelerators and brakes in
the pathogenesis of cerebral malaria. Trends Immunol. 24,
491–499.
Hunt, N.H., Golenser, J., Chan-Ling, T., Parekh, S., Rae, C., Potter, S.,
Medana, I.M., Miu, J., Ball, H.J., 2006. Immunopathogenesis of
cerebral malaria. Int. J. Parasitol. 36, 569–582.
Hunter, C.A., Jennings, F.W., Kennedy, P.G.E., Murray, M., 1992a.
Astrocyte activation correlates with cytokine production in
central nervous system of Trypanosoma brucei brucei-infected
mice. Lab. Investig. 67, 635–642.
Hunter, C.A., Jennings, F.W., Kennedy, P.G.E., Murray, M., 1992b.
The use of azathioprine to ameliorate post-treatment
encephalopathy associated with African trypanosomiasis
Neuropathol. Appl. Neurobiol. 18, 619–625.
Ilieva, H., Polymenidou, M., Cleveland, D.W., 2009. Non-cell
171
autonomous toxicity in neurodegenerative disorders: ALS and
beyond. J. Cell Biol. 187, 761–772.
John, C.C., Bangirana, P., Byarugaba, J., Opoka, R.O., Idro, R., Jurek,
A.M., Wu, B., Boivin, M.J., 2008. Cerebral malaria in children is
associated with long-term cognitive impairment. Pediatrics
122, e92–e99. doi:10.1542/peds.2007-3709 .
Kashyap, A., Anand, K., Kashyap, S., 2004. Correspondence—rabies
and other lyssavirus disease. Lancet 363, 1906.
Keita, M., Bouteille, B., Enanga, B., Vallat, J.M., Dumas, M., 1997.
Trypanosoma brucei brucei: a long-term model of human African
trypanosomiasis in mice, meningo-encephalitis, astrocytosis,
and neurological disorders. Exp. Parasitol. 85, 183–1992.
Kennedy, P.G.E., 2007. The Fatal Sleep. Luath Press Ltd, Edinburgh.
Kennedy, P.G.E., 2008. The continuing problem of human African
trypanosomiasis (sleeping sickness). Ann. Neurol. 64,
116–127.
Kennedy, P.G.E., 2009. Cytokines in central nervous system
trypanosomiasis: cause, effect or both? Trans. R. Soc. Trop.
Med. Hyg. 103, 213–214.
Kettenmann, H., Verkhratsky, A., 2008. Neuroglia: the 150 years
after. Trends Neurosci. 31, 653–659.
Knobel, D.L., Cleaveland, S., Coleman, P.G., Fèvre, E.M., Meltzer, M.
I., Miranda, M.E.G., Shaw, A., Zinsstag, J., Meslin, F.-X., 2005.
Re-evaluating the burden of rabies in Africa and Asia. Bull.
World Health Organ. 83, 360–368.
Kristensson, K., Dastur, D.K., Manghani, D.K., Tsiang, H.,
Bentivoglio, M., 1996. Rabies: interactions between neurons
and viruses. A review history Negri inclusion bodies
Neuropathol. Appl. Neurobiol. 22, 179–187.
Kristensson, K., Nygård, M., Bertini, G., Bentivoglio, M., 2010.
African trypanosome infections of the nervous system:
parasite entry and effects on neuronal functions. Prog.
Kuang, Y., Lackay, S.N., Zhao, L., Fu, Z.F., 2009. Role of chemokines
in the enhancement of BBB permeability and inflammatory
infiltration after rabies virus infection. Virus Res. 144, 18–26.
Lafon, M., 2005. Rabies virus receptors. J. Neurovirol. 11, 82–87.
Lafon, M., 2008. Immune evasion, a critical strategy for rabies
virus. Dev. Biol. 131, 403–409.
Lahaye, X., Vidy, A., Pomier, C., Obiang, L., Harper, F., Gaudin, Y.,
Blondel, D., 2009. Functional characterization of Negri bodies
(NBs) in rabies virus-infected cells: evidence that NBs are sites
of viral transcription and replication. J. Virol. 83, 7948–7958.
Laothamatas, J., Wacharapluesadee, S., Lumlertdacha, B.,
Ampawong, S., Tepsumethanon, V., Shuangshoti, S.,
Phumesin, P., Asavaphatiboon, S., Worapruekjaru, L.,
Avihingsanon, Y., Israsena, N., Lafon, M., Wilde, H.,
Hemachudha, T., 2008. Furious and paralytic rabies of canine
origin: neuroimaging with virological and cytokine studies. J.
Neurovirol. 14, 119–129.
Laveran, A., 1881. Un nouveau parasite trouvé dans le sang de
malades atteints de fièvre palustre. Origine parasitaire des
accidents de l’impaludisme. Bull. Mém. Soc. Méd. Hopitaux
Paris 17, 158–164.
Laveran, A., 1893. Paludism. New Sydenhan Society, London.
Levine, J.B., Kong, J., Nadler, M., Xu, Z.S., 1999. Astrocytes interact
intimately with degenerating motor neurons in mouse
amyotrophic lateral sclerosis (ALS). Glia 28, 215–224.
Li, M., Ona, V.O., Guégan, C., Chen, M., Jackson-Lewis, V., Andrews, L.
J., Olszewki, A.J., Stieg, P.E., Lee, J.P., Przedborski, S.,
Friedlander, R.M., 2000. Functional role of caspase-1 and
caspase-3 in an ALS transgenic mouse model. Science 288,
335–339.
Lino, M.M., Schneider, C., Caroni, P., 2002. Accumulation of SOD1
mutants in postnatal motoneurons does not cause
motoneuron pathology or motoneuron disease. J. Neurosci. 22,
4825–4832.
Lundkvist, G.B., Kristensson, K., Bentivoglio, M., 2004. Why
172 B RA I N R E SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3
trypanosomes cause sleeping sickness. Physiology 19,
198–206.
Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., Locati,
M., 2004. The chemokine system in diverse forms of
macrophage activation and polarization. Trends Immunol. 25,
677–686.
Marchiafava, E., Bignami, A., 1892. Sulle febbri malariche
estivo-autunnali. Bollettino della Regia Accademia Medica di
Roma 18, 1-169. English translation: on summer-autumnal
malaria fevers. Malaria and the Parasites of Malaria Fevers,
Vol 150. The New Sydenham Society, London, pp. 1–234.
Marchiafava, E., Celli, A., 1887. Sull'infezione malarica. Bollettino
Regia Accademia Medica di Roma 18.
Margreth, A., 2003. Adelchi Negri and schools of General Pathology
in Italy between the end of the nineteenth and beginning of the
twentieth century. Rend. Fis. Acc. Lincei 14, 251–262.
Malerczyk, C., Nel, L.H., Gniel, D., Blumberg, L., 2010. Rabies in
South Africa and the FIFA Soccer World Cup: travelers'
awareness for an endemic but neglected disease. Hum. Vaccin.
6 (Epub ahead of print May 5).
Masocha, W., 2010. CD200 receptors are differentially expressed
and modulated by minocycline in the brain during
Trypanosoma brucei infection. J. Neuroimmunol. 226, 59–65.
Masocha, W., Robertson, B., Rottenberg, M.E., Mhlanga, J., Sorokin, L.,
Kristensson, K., 2004. Cerebral vessel laminins and IFN-γ define
Trypanosoma brucei brucei penetration of the blood–brain barrier.
J. Clin. Investig. 5, 689–694.
Masocha, W., Rottenberg, M.E., Kristensson, K., 2007. Migration of
African trypanosomes across the blood–brain barrier. Physiol.
Behav. 92, 110–114.
Masocha, W., Amin, D.N., Kristensson, K., Rottenberg, M.E., 2008.
Differential invasion of Trypanosoma brucei brucei and
lymphocytes into the brain of C57BL/6 and 129Sv/EV mice.
Scand. J. Immunol. 68, 484–491.
Mazzarello, P., 2010. Golgi. Oxford University Press, Oxford.
Matteoli, M., Verderio, C., 2011. ATP in neuron-glia bidirectional
signalling. Brain Res. Rev 66, 106–114.
Medana, I.M., Turner, G.D.H., 2006. Human cerebral malaria and
the blood–brain barrier. Int. J. Parasitol. 36, 555–568.
Medana, I.M., Chan-Ling, T., Hunt, N.H., 2000. Reactive changes of
retinal microglia during fatal murine cerebral malaria: effects
of dexamethasone and experimental permeabilization of the
blood–brain barrier. Am. J. Pathol. 156, 1055–1065.
Medana, I.M., Chauduri, G., Chan-Ling, T., Hunt, N.H., 2001. Central
nervous system in cerebral malaria: ‘innocent bystander’ or
active participant in the induction of immunopathology?
Immunol. Cell Biol. 79, 101–120.
Medana, I.M., Idro, R., Newton, C.R.J.C., 2007. Axonal and astrocyte
injury markers in the cerebrospinal fluid of Kenyan children
with severe malaria. J. Neurol. Sci. 258, 93–98.
Medawar, P.B., 1948. Immunity to homologous grafted skin. III.
The fate of skin homografts transplanted to the brain, to
subcutaneous tissue, and to the anterior chamber of eye. Br. J.
Exp. Pathol. 29, 58–69.
Medzhitov, R., 2008. Origin and physiological roles of inflammation.
Nature 454, 428–435.
Ménager, P., Roux, P., Mégret, F., Bourgeois, J.-P., Le Sourd, A.-M.,
Danckaert, A., Lafage, M., Préhaud, C., Lafon, M., 2009. Toll-like
receptor 3 (TLR3) plays a major role in the formation of rabies
virus Negri bodies. PLoS Pathog. 5 (2), e10000315. doi:10.1371/
journal.ppat.1000315 . Santamaria, L., 1994. Camillo Golgi as clinical pathologist:
Mhlanga, J.D.M., Bentivoglio, M., Kristensson, K., 1997. Neurobiology
of cerebral malaria and African sleeping sickness. Brain Res. Bull.
44, 579–589.
Mitrabhakdi, E., Shuangshoti, S., Wannakroit, P., Lewis, R.A.,
Susuki, K., Laothamatas, J., Hemachudha, T., 2005. Difference
in neuropathogenetic mechanisms in human furious and
paralytic rabies. J. Neurol. Sci. 238, 3–10.
Miyamoto, K., Matsumoto, S., 1967. Comparative studies between
pathogens of street and fixed rabies infection. J. Exp. Med. 125,
447–456.
Mott, F.W., 1907. Histological observations on the changes in the
nervous system in trypanosome infections especially sleeping
sickness and their relation to syphilitic lesions of the nervous
system. Arch. Neurol. 3, 581–646.
Mulenga, C., Mhlanga, J.D.M., Kristensson, K., Robertson, B., 2001.
Trypanosoma brucei brucei crosses the blood–brain barrier while
tight junction proteins are preserved in a rat chronic disease
model. Neuropathol. Appl. Neurobiol. 27, 77–85.
Muscatello, U., 2007. Golgi's contribution to medicine. Brain Res.
Rev. 55, 3–7.
Nakamichi, K., Saiki, M., Sawada, M., Takayama-Ito, M., Yamamuro,
Y., Morimoto, K., Kurane, I., 2005. Rabies virus-induced activation
of miogeno-activated protein kinase and NF-kB signaling
pathways regulates expression of CXC and CC chemokine ligands
in microglia. J. Virol. 179, 11801–11812.
Negri, A., 1903. Contributo allo studio dell'eziologia della rabbia.
Boll. Soc. Med. Chir. Pavia 2, 88–115.
Negri Luzzani, L., 1913. Le diagnostic de la rage par la
démonstration du parasite spécifique. Résultats de dix ans
d'expériences. Ann. Inst. Pasteur 27, 1039–1064.
Neumann, H., Kotter, M.R., Franklin, R.J.M., 2009. Debris clearance
by microglia: an essential link between degeneration and
regeneration. Brain 132, 288–295.
Newton, C.R.J.C., 2000. Cerebral malaria. J. Neurol. Neurosurg.
Psychiatry 69, 433–441.
Nigg, A.J., Walker, P.L., 2009. Overview, prevention, and treatment
of rabies. Pharmacotherapy 29, 1182–1195.
Okun, E., Griffioen, K.J., Lathia, J.D., Tang, S.-C., Mattson, M.P.,
Arumugam, T.V., 2009. Toll-like receptors in neurodegeneration.
Brain Res. Rev. 59, 278–292.
Pasteur, L., 1885. Méthode pour prevenir la rage après morsure. CR
Acad. Sci. 101, 765–773.
Pierce, S.K., Miller, L.H., 2009. World malaria day 2009: what
malaria knows about the immune system that immunologists
still do not. J. Immunol. 182, 5171–5177.
Prosniak, M., Hooper, D.C., Dietzschold, B., Koprowski, H., 2001.
Effect of rabies virus infection on gene expression in mouse
brain. Proc. Natl. Acad. Sci. USA 98, 2758–2763.
Quan, N., Mhlanga, J.D.M., Whiteside, B., McCoy, A.N., Kristensson, K.,
Herkenham, M., 1999. Chronic overexpression of proinflammatory
cytokines and histopathology in the brains of rats infected with
Trypanosoma brucei. J. Comp. Neurol. 414, 114–130.
Quan, N., He, L., Lai, W., 2003. Intraventricular infusion of
antagonists of IL-1 and TNFα attenuates neurodegeneration
induced by the infection of Trypanosoma brucei.
J. Neuroimmunol. 138, 92–98.
Rasti, N., Wahlgren, M., Chen, Q., 2004. Molecular aspects of
malaria pathogenesis. FEMS Immunol. Med. Microbiol. 41, 9–26.
Ray, N.B., Power, C., Lynch, W.P., Ewalt, L.C., Lodmell, D.L., 1997.
Rabies viruses infect primary cultures of murine, feline,
and human microglia and astrocytes. Arch. Virol. 142, 1011–1019.
Rivest, S., 2009. Regulation of innate immune responses in the
brain. Nat. Rev. Immunol. 9, 429–439.
Rock, R.B., Gekker, G., Xu, S., Sheng, W.S., Cheeran, M., Lokensgard, J.R.,
Peterson, P.K., 2004. Role of microglia in central nervous system
infections. Clin. Microbiol. Rev. 17, 942–964.
Rupprecht, C.E., Hanlon, C.A., Hemachudha, T., 2002. Rabies
re-examined. Lancet Infect. Dis. 2, 327–343.
epicritical reading of Golgi's works on malaria. Med. Secoli Arte
Sci. 6, 581–608.
Schnell, M.J., McGettigan, J.P., Wirblich, C., Papaneri, A., 2010. The
cell biology of rabies virus: using stealth to reach the brain. Nat.
Rev. Microbiol. 8, 51-41.
Scott, C.A., Rossiter, J.P., Andrew, R.D., Jackson, A.C., 2008.
Structural abnormalities in neurons are sufficient to explain
the clinical disease and fatal outcome of experimental rabies
 B RA I N RE SE A R CH RE V I EW S 66 ( 20 1 1 ) 1 5 2– 1 7 3 173

in yellow fluorescent protein-expressing transgenic mice. imported into Japan from the Philippines: immunohisto-
J. Virol. 82, 513–521. chemistry. Pathol. Int. 59, 555–566.
Sherwood, J., 1999. Syphilization: human experimentation in the Tognotti, E., 2007. Camillo Golgi e il contributo di scienziati
search for a syphilis vaccine in the nineteenth century. J. Hist. italiani allo sviluppo della malariologia nell'ultimo quarto del
Med. 54, 364–386. diciannovesimo secolo. Med. Secoli 19, 101–117.
Simarro, P.P., Jannin, J., Cattand, P., 2008. Eliminating human Turner, G., 1997. Cerebral malaria. Brain Pathol. 7, 569–582.
African trypanosomiasis: where do we stand and what comes Ugolini, G., 1995. Specificity of rabies virus as a transneuronal
next? PLoS Med. 5 (2), e55. doi:10.1371/journal.pmed.0050055 . tracer of motor networks: transfer from hypoglossal moto-
Sofroniew, M.V., Vinters, H.V., 2010. Astrocytes: biology and neurons to connected second-order and higher order central
pathology. Acta Neuropathol. 119, 7–35. nervous system cell groups. J. Comp. Neurol. 356, 457–480.
Song, M., Tang, Q., Wang, D.-M., Mo, Z.-J., Guo, S.-H., Li, H., Tao, X.-Y., Ugolini, G., in press. Advances in viral transneuronal tracing. J.
Rupprecht, C.E., Feng, Z.-J., Liang, G.-D., 2009. Epidemiological Neurosci. Meth. 194,2–20.
investigations of human rabies in China. BMC Infect. Dis. 9, 210. Verkhratsky, A., Parpura, V., Rodriguez, J.J., 2011. Where the
doi:10.1186/1471-2334-9-210 . thoughts dwell: the physiology of neuronal–glial “diffuse
Spielmeyer, W., 1908. Die Trypanosomenkrankheiten und ihre neural net”. Brain Res. Rev 66, 133–151.
Beziehungen zu den syphilogenen Nervenkrankheiten. Vinson, C.R., Adler, P.N., 1987. Directional non-cell autonomy and
Fischer, Jena. the transmission of polarity information by the frizzled gene of
Sterverding, D., 2008. The history of African trypanosomiasis. Drosophila. Nature 329, 549–551.
Parasites Vectors 1, 3. doi:10.1186/1756-3305-1-3 . Walter, L., Neumann, H., 2009. Role of microglia in neuronal
Streit, W.J., Mrak, R.E., Griffin, W.S.T., 2004. Microglia and degeneration and regeneration. Semin. Immunopathol. 31,
neuroinflammation: a pathological perspective. 513–525.
J. Neuroinflamm. 1, 14. doi:10.1186/1742-2094-1-14 . White, N.J., Turner, G.D.H., Medana, I.M., Dondorp, A.M., Day, N.P.J.,
Suja, M.S., Mahadevan, A., Madhusudhana, S.N., Vijayasarathi, S.K., 2009. The murine cerebral malaria phenomenon. Trends Parasitol.
Shanakar, S.K., 2009. Neuroanatomical mapping of 26, 11–15.
rabies nucleocapsid viral antigen distribution and Wilson, E.H., Weninger, W., Hunter, C.A., 2010. Trafficking of
apoptosis in pathogenesis in street dog rabies—an immune cells in the central nervous system. J. Clin. Investig.
immunohistochemical study. Clin. Neuropathol. 28, 113–124. 120, 1368–1379.
Szklarczyk, A., Stins, M., Milward, E.A., Ryu, H., Fitzsimmons, C., Wunner, W.H., Briggs, D.J., 2010. Rabies in the 21st century
Sullivan, D., Connat, K., 2007. Glial activation and matrix PLoS Negl. Trop. Dis. 4 (3), e591. doi:10.1371/journal.
metalloproteinase release in cerebral malaria. J. Neurovirol. pntd.0000591 .
13, 2–10. Zhao, W., Xie, W., Le, W., Beers, D.R., He, Y., Henkel, J.S., Simpson, E.P.,
Tepper, R.I., Pattengale, P.K., Leder, P., 1989. Murine interleukin- Yen, A.A., Xiao, Q., Appel, S.H., 2004. Activated
4 displays potent anti-tumor activity in vivo. Cell 57, microglia initiate motor neuron injury by nitric oxide and
503–512. glutamate-mediated mechanism. J. Neurophathol.
Tobiume, M., Sato, Y., Katano, H., Nakajima, N., Tanaka, K., Exp. Neurol. 63, 964–977.
Noguchi, A., Inoue, S., Hasegawa, H., Iwasa, Y., Tanaka, J., Zhao, L., Toriumi, H., Kuang, Y., Chen, H., Fu, Z.F., 2009. The role of
Hayashi, H., Yoshida, S., Kurane, I., Sata, T., 2009. Rabies virus chemokines in rabies virus infection: overexpression may not
dissemination in neural tissues of autopsy cases due to rabies always be beneficial. J. Virol. 83, 11808–11818.