Acta Neurochir (2000) [Suppl) 76: 535-539
© Springer-Verlag 2000
Blockade of Histamine H2 Receptors Attenuate Blood-Brain Barrier Permeability,
Cerebral Blood Flow Disturbances, Edema Formation and Cell Reactions
Following Hyperthermic Brain Injury in the Rat
R. Patnaik l •2 , S. Mohanty\ and H. S. Sharma l
1 Laboratory of Neuroanatomy. Department of Medical Cell Biology, Biomedical Centre, Uppsala University, Uppsala. Sweden
2 Department of Biomedical Engineering, Institute of Technology, Banaras Hindu University, Varanasi, India
3 Departrnent of Neurosurgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
Summary
Role of histamine H2 receptors in blood-brain barrier (BBB) dis-
turbances, cerebral blood flow (CBF), brain edema formation, and
cell injury caused by heat stress in a rat model was examined using
the pharmacological approach. Blockade of histamine H2 receptors
by cimetidine or ranitidine significantly attenuated the BBB perme-
ability to Evans blue albumin and 1l31 II-sodium extravasation, brain
edema formation and cell injury following 4 h heat stress in rats at
38 DC. These drug treatments also restored the CBF to near normal
values. These beneficial effects in heat stress were most marked in
rats treated with ranitidine compared to cimetidine given in identical
dosage. Our observations suggest that blockade of histamine H2 re-
ceptor is beneficial in hyperthermic brain injury and indicates that
histamine is involved in the pathophysiology of heat stress induced
brain dysfunction. Our study strongly suggests further need to de-
velop more specific and sensitive histaminergic H2 receptor blockers
for the treatment of neurological ailments.
Keywords: Histamine; brain edema; histamine H2 receptor antag-
onists; cerebral blood flow.
Introduction
The mechanisms behind breakdown of the blood-
brain barrier (BBB) in hyperthermia is not well un-
derstood [5, 14, 16, 17]. The BBB is a physiologically
dynamic barrier [1,3]. The physicochemical properties
of the barrier is very similar to that of an extended
plasma membrane [3, 4]. The BBB mainly resides
within the endothelial cell of cerebral vessels which are
connected with tight junctions [1, 3, 4, 17]. The cere-
bral endothelial cells also lack vesicular transport
[3, 4]. The BBB remains very tight under normal con-
ditions [1, 16, 17]. However, in several pathological
conditions, the BBB becomes leaky and extravasation
of serum proteins will take place into the brain extra-
cellular compartment [1, 3, 4, 16, 17] resulting in
vasogenic edema and adverse cell reaction [4, 14].
The cerebral endothelium contains receptors of
several neurochemicals which are known mediators of
the BBB permeability and brain edema formation
[1,4, 11]. Release of several neurochemicals occur in
hyperthermia which may act on the cerebral endothe-
lium to induce a receptor mediated breakdown of the
BBB permeability and consequently cerebral edema
formation [14, 16, 17]. Previous reports from our
laboratory suggest that serotonin, prostaglandin and
opioids are involved in the mechanisms of BBB
breakdown in hyperthermic injury [14]. However,
apart from these neurochemicals histamine is also
liberated from mast cells or histaminergic neurons
which can also influence the BBB function [10, 18].
Both histamine HI and histamine H2 receptors are
located within the endothelium [11] and histamine H2
receptors are known to be involved in the BBB dis-
ruption following trauma or intracarotid histamine
infusion [9, 11-13].
Histamine is a neurotransmitter in the central
nervous system (CNS) and histaminergic neurons are
present in large numbers in the brain [18]. The pro-
jections of histaminergic neurons are distributed
throughout the CNS [4, 18]. In spite of a dense and
widespread presence of histaminergic neurons, there
are only few previous reports which indicate that the
amine is involved in the central mechanisms of ther-
moregulation [2]. However, its involvement in hyper-
thermic brain injury is still not well known. Traumatic
injuries to the brain will induce an increase in plasma
536
and brain histamine levels and the amine is involved in
the pathogenesis of brain edema in various traumatic
or hypoxic injuries of the CNS, probably via histamine
H2 receptors [10]. Since hyperthermia has the capacity
to induce leakage of the BBB [17], a possibility exists
that endogenous histamine will contribute to some of
these changes [13].
The present investigation was carried out to under-
stand the involvement of histamine in the pathophysi-
ology of hyperthermic brain injury by blockade of
histamine H2 receptors using two potent H2 receptor
antagonist cimetidine and ranitidine [8, 9]. Since his-
tamine is known to influence vasomotor tone of the
cerebral microvessels, influence of histamine H2 re-
ceptor antagonists on the CBF was also examined.
Materials and Methods
Animals
Experiments were carried out on male Wistar rats (90-100 g, age
8-9 weeks) housed at controlled room temperature (21 ± 1°C) with
12 h light and 12 h dark schedule. Rat feed and tap water were sup-
plied ad libitum before the experiments.
Heat Exposure
Animals were exposed to heat stress in a biological oxygen demand
incubator (BOD, wind velocity 20-26 em/sec; relative humidity 50-
55%) maintained at 38°C for 4 h [12). This experimental condition is
approved by the Ethical Committee ofUppsala University, Uppsala,
Sweden; Lund University, Lund, Sweden and Banaras Hindu Uni-
versity, Varanasi, India. Rats kept at room temperature were used as
controls.
Treatment with Cimetidine and Ranitidine
In separate groups of rats, two prototypes of histamine H2 recep-
tor antagonists, cimetidine or ranitidine (Sigma Chemical Co., USA)
were administered (10 mg/kg, i.p.) 30 min before the onset of heat
stress [12, 16, 17).
Stress Symptoms
In all animals subjected to heat stress, the rectal temperature, be-
havioural salivation, prostration and occurrence of gastric ulceration
in the stomach was measured as indices of stress response [17).
Blood-Brain Barrier Permeability
The blood-brain barrier (BBB) permeability was measured using
Evans blue albumin and [1311I-sodium [16, 17) according to the stan-
dard protocol.
Cerebral Blood Flow
The CBF was measured using carbonised microsphere (15 ±
0.6 ~ diameter) labelled with [1251 Iodine as described earlier [12, 13).
R. Patnaik et al.
Brain Edema
The water content of the brain was measured to determine brain
edema [4). For this purpose brain samples were taken and weighed
immediately. Thereafter the wet samples were placed in an oven
maintained at 90°C for 72 h to obtain dry weight of these samples.
The brain water content was calculated from the differences in the
dry and wet weight of the samples.
Cell Injury
In separate groups of rats cell injury in the cerebral cortex
was determined using standard light and electron microscopy as
described earlier [14).
Statistical Analysis
Student's unpaired t-test was used to evaluate the statistical sig-
nificance of the data obtained. A p-value less than 0.05 was consid-
ered to be significant.
Results
Effect of Cimetidine and Ranitidine on Stress
Symptoms
Pretreatment with cimetidine or ranitidine did not
influence stress symptoms in general. However, a mild
but significant reduction in hyperthermia was evident
in these drug treated rats compared to the untreated
stressed group (Fig. 1). The occurrence of gastric hae-
morrhages, was significantly attenuated by cimetidine
and ranitidine treatment. The drug treatments alone,
however, did not influence these parameters in normal
rats.
Effect of Cimetidine and Ranitidine on the BBB
Permeability
Subjection of rats to 4 h heat stress resulted in a
marked increase in the permeability of Evans blue and
radioactive iodine in several brain regions. Pretreat-
ment with cimetidine or ranitidine significantly reduced
the extravasation of these tracers in the brain following
heat stress (Fig. 1). The effect of ranitidine was most
pronounced in attenuating the BBB permeability to
these tracers in heat stress.
Effect of Cimetidine and Ranitidine on the Brain
Edema Formation
Pretreatment with ranitidine or cimetidine signifi-
cantly attenuated brain edema formation following
4 h heat exposure in rats (Fig. 1). This effect was most
Blockade of Histamine H2 Receptors 537

4
•• 1311-sodlum %
Evans Blue mg%

0
control 4hHS cimetidlne ranitidine
+HS +HS

1.6 CBF mllg/min

85 Brain Water %

**
1.4 83

1.2 81

1.0 79

0.8 77

0.6 75
control 4h HS cimetidlne ranitidine control 4h HS cimetidine ranhid lne
+ HS + HS + HS +HS

Fig. I. Effects of cimetidine and ranitidine on heat stress induced changes in rectal temperature (upper panel, left); blood-brain barrier (BBB)
permeability (upper panel, right); cerebral blood flow (eBF) (lower panel, left) and brain water content (lower panel right). • P < 0.05;
** = P < 0.01 compared to control; ~~ = P < 0.01 compared to 4 h heat stress (HS); Student's unpaired t-test. Values are mean ± SD

pronounced in rats which received ranitidine as pre-
treatment compared to the cimetidine treatment. Nor-
mal rats received ranitidine or cimetidine, however,
did not exhibit any significant alterations in the brain
water content.
distortion of the nerve cells, swollen glial cells, mem-
brane disruption, edema and myelin vesiculation were
quite frequent in untreated heat stressed rats. Pre-
treatment with ranitidine or cimetidine markedly re-
duced cell injury following heat stress.

Effect of Cimetidine and Ranitidine on CBF Discussion

At the end of 4 h heat stress, the CBF declined by
30% from the control value (Fig. 1). Pretreatment with
cimetidine or ranitidine significantly attenuated ische-
mia caused by heat stress. Thus the CBF was restored
near normal values in drug treated heat stressed
animals. Ranitidine seems to be more effective in re-
storing CBF values compared to cimetidine. On the
other hand, treatment of ranitidine or cimetidine in
normal rats did not influence the CBF significantly.
Effect of Cimetidine and Ranitidine on Cell Injury
Heat stress induces marked neuronal, glial and
axonal changes in the cerebral cortex of rats. Thus,
These present results show that blockade of hista-
mine H2 receptors seems to be beneficial in the patho-
physiology of brain injury caused by heat stress. This
indicates the use of histamine H2 receptor antagonists
as a suitable therapeutic tool in neurological diseases,
a feature which however, requires additional investi-
gation. The salient new findings of the present investi-
gation further show that the effects of ranitidine given
in identical doses is far superior than cimetidine in
attenuating heat stress induced hyperthermia, BBB
dysfunction, brain edema, CBF changes and cell in-
jury. This observation suggests that ranitidine is more
effective than cimetidine. Previously, ranitidine pre-
treatment significantly attenuated hypertension in-
538
duced breakdown of the BBB permeability [9]. Our
results for the first time show that ranitidine is also
capable to reduce microvascular permeability distur-
bances in heat stress. The probable mechanisms be-
hind superior effects of ranitidine over cimetidine is not
known. It seems likely that ranitidine is more specific
to histamine H2 receptors and may cross the normal
BBB more effectively than cimetidine [8]. However,
further research is needed to explore this point.
Involvement of histamine in the mechanisms of
thermoregulation is not well known. It seems likely
that histamine is released from cerebral mast cells,
histaminergic nerve fibres or from other peripheral
sources [19]. An increased level of histamine in heat
stress will induce a breakdown of the BBB permeabil-
ity probably by stimulating histamine H2 receptors. A
breakdown of the BBB permeability will contribute to
vasogenic edema formation which in tum may induce
cell injury. Increased histamine concentrations in the
plasma or brain may act on local microvessels to in-
fluence the local cerebral blood flow [18]. Since hista-
mine in large doses mainly accounts for vasoconstric-
tion, a reduction in the CBF is quite likely following
histamine elevation in the circulation or its release
through various sources.
The above hypothesis is well supported by our ob-
servations using two potent histamine H2 receptor
antagonists cimetidine and ranitidine. Cimetidine is
known to reduce brain edema caused by a focal inci-
sion of the cerebral cortex [10]. Thus, the mechanisms
of brain edema formation following a focal lesion
or hyperthermia appears quite similar to that of brain
injury. In the brain injury model, a significant increase
in plasma and brain histamine was found following 5 h
after lesion, indicating that trauma has the capacity to
induce histamine increase in the brain and plasma [10].
It may be that histamine level in circulation will also
increase in hyperthermia. To further confirm this hy-
pothesis, measurements of the plasma and brain hista-
mine in heat stress are needed.
Histamine has the capacity to induce brain edema
[11]. Thus, intracarotid histamine infusion induces
brain edema and disrupts the BBB permeability [18].
Blockade of histamine H2 receptors with high doses of
cimetidine reduces irradiation induced brain edema
indicating involvement of histamine [9-11]. Our ob-
servations with histamine H2 receptor antagonists in
heat stress are in good agreement with these findings
and further show that histamine H2 receptor antago-
nists are also able to reduce the magnitude of cell in-
R. Patnaik et al.
jury caused by hyperthermia. A reduction in cell injury
by blockade of histamine H2 receptors may be due
to the ability of a reduction in the BBB permeability,
cerebral blood flow and brain edema. A reduction of
these factors alone or in combination will result in sig-
nificant neuroprotection [17].
Furthermore, our observations suggest that hista-
mine H2 receptor antagonists attenuate hyperthermia
following heat stress. A reduction in the magnitude of
hyperthermia may induce less release of neurochemi-
cals or histamine thereby influencing neuroprotection.
A significantly less rise in the body temperature fol-
lowing heat stress in drug treated rats clearly suggests
that the amine is involved in the pharmacology of
thermoregulation at high ambient temperature. How-
ever, further research is needed to clarify this point.
Histamine may also exert some direct effects on the
cerebral endothelial cells to influence nitric oxide for-
mation probably via histamine H2 receptors. Thus, ci-
metidine and ranitidine inhibits nitric oxide induced
endothelial relaxation in the cerebrovascular bed [6].
Since nitric oxide is a potential contributor of the BBB
breakdown, brain edema and cell injury, blockade of
NO by these drug treatments may also induce neuro-
protection [17]. To further confirm this point, NOS
upregulation in cimetidine or ranitidine treated ani-
mals in heat stress are needed.
In conclusion, our observations suggest that hista-
mine is involved in the pathophysiology of the BBB
permeability, brain edema formation and cerebral cir-
culatory disturbances in heat stress and histamine H2
receptors play an important role in the pathophysiol-
ogy of hyperthermic brain injury.
Acknowledgments
This investigation is supported by grants from Swedish Medical
Research Council nrs. 02710 (JW, HSS); Garan Gustafsson Stiftelse
(HSS), Sweden; and The University Grants Commission (HSS),
New Delhi, India. The technical assistance of Kiirstin Flink, Kerstin
Rystedt, Inga Harte, Una Johansson and Secretarial assistance of
Gun-Britt Lind and Aruna Sharma are highly appreciated with
thanks.
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Correspondence: Hari Shanker Sharma, Ph.D., Laboratory of
Neuroanatomy, Department of Medical Cell Biology, Box 571,
Biomedical Centre, Uppsala University, S-75123 Uppsala, Sweden.