Chemical anatomy of pain pathways Primary afferent nociceptors are specialised free nerve endings of primary afferents A &

; C fibres. They detect noxious stimuli anything that has produced tissue damage or threatens to do so. y A fibres o Myelinated fast conduction velocity -> sharp, pricking, fast (epicritic) pain informative (precise location of insult) --> reflex withdrawal o Activated by mechanical/thermal stimuli (high threshold)

Not all A fibres are noxious some are Down Hair receptors

o 2 main classes of A fibres -> can be distinguished by their differential responses to heat o A fibres also exist which are propr ioceptors in muscle spindles & A fibres which are low threshold mechanoceptors but 20% are high threshold so can serve as nociceptors


y C fibres

Both types innervate specific peripheral receptors

o Unmyelinated slow conduction velocity -> slow, burning (protopathic) pain --> changes behaviour of person & emotional reaction o Polymodal (activated by mechanical, thermal & chemical stimuli) o Not all C-fibres are nociceptors there are also some unmyelinated low threshold mechanoreceptors Cell bodies of the nociceptors are located in the dorsal root ganglion. They are small. Small DRG cells can be divided as:

 Myelinated (A ) contain neurofilament NF200  Unmyelinated (C) petidergic; contain substance P, CGRP &somatostatin
o Start in stratum spinosum& ends in lamina I and lamina II outer o Express TrkA -> depend on NGF for survival

 Unmyelinated (C) non peptidergic; contain FRAP & cell surface

glycoproteins which bind the plant lectin IB4 o Start in stratum granulosum& ends in lamina II inner

o Mass related gene coupled protein receptors distinguishes subtypes o Express GFR 1/2 & receptor tyrosine kinase Ref -> depend on GDNF for survival o Also express P2X3 receptors Cell marker correlates with peripheral target tissue innervated too:

 Visceral ->peptidergic  Skin -> mostly peptidergic  Muscle -> small % non peptidergic
Sensory transduction: noxious stimuli detected -> transduced into inward currents that if large enough generates APs along axon to CNS Ion channels expressed in axon terminals and ce ll soma when activated lead to inward currents that depolarise the axon (generator potential) -> if large enough APs fires. Lots of different types: TRP opens in respond to heat, H +&capsacin o TRPV1 expressed by 60% of peptidergic& 75% of non -peptidergic neurones P2X3 Na + channels i.e. 1.8, 1.9 Target tissue --> peripheral nerve --> DRG (soma) --> dorsal root --> spinal cord Cell bodies with the largest diameters give rise to myelinated, rapidly conducting A fibres ->most detect innocuous stimuli but 20% are HTMs. By contrast, small & medium diameter cell bodies give rise to most of the nociceptors. Dorsal horn can be divided into 6 parallel laminae patterns of termination of primary afferents within the spinal cord related to axonal diameter and receptive field modality. Superficial dorsal horn (I -III) Main target for nociceptive primary afferents Lamina II can be divided into IIi&IIo

Remaining 3 laminae for innocuous stimuli but nociceptive afferents also terminate here Lamina I (marginal layer) projection neurones & interneurons y Projection neurones larger than interneurons (esp. marginal cells of Waldeyer) Lamina II (substantiagelatinosa lack of myelinated fibres) interneurons y Most of the interneurons packed into IIo o Can differentiate between IIo& Iii due to lack of myelin in Iii as there are no interneurons Lamina III high density of interneurons (but bigger than those in lamina II) Lamina IV-VI heterogenous with neurones of different sizes including projection neurones (esp. V) Afferent fibres entering through dorsal root divide into ascending and descending branches; mostly terminate near point of entry but can travel several segments up/down in Lissauers tract. Primary afferents release primarily glutamate onto neurones within the dorsal horn including:   Projection axons convey info to the brain Interneurones remain in spinal cord and serve 3 major functions: o Relay sensory input from the periphery that may modulate motor output i.e. reflexes o Relay as well as modulate descending pathways

Excitatory: glutamate (stalked VGlut2/PKC )

 High threshold noxious stimuli only  Low threshold innocuous stimuli only  Wide dynamic range noxious & innocuous stimuli 
Inhibitory: GABA, glycine (islet GABA)

A fibres activate the dorsal column medial lemniscus

DRG -> dorsal column ->cuneate/gracile nucleus -> internal arcuate (decussating) fibres -> medial lemniscus -> VPL thalamus -> somatosensory cortex A fibres activate the spinothalamic tract -> fast pain Ventral white commissure (decussate straight away) -> spinal lemniscus -> VPL thalamus -> somatosensory cortex C fibres activate the spinoreticular tract -> slow pain Neurochemistry of dorsal horn interneurons: Inhibitory o GABAergic but not glycinergic

     

Neuropeptides: NPY, galanin, encephalin Miscellaneous: NOS Miscellaneous: parvalbumin, NOS Neuropeptides: somatostatin, neurotensin Miscellaneous: calbindin, PKC Receptors: , NK1

o GABAergic but glycinergic Excitatory (VGlut2)

Projection neurones: expre ss NK1 receptor which binds substance P Descending monoaminergic axons: Serotonin from medullary Raphe nuclei Noradrenaline from locus coerulus Modulation by interneurons: intrathecal administration of GABA A and glycine receptor antagonist can cause allo dynia (innocuous stimuli causes nociception i.e. brushing of skin) -> suggests inhibitory interneurons supress activity evoked by tactile afferents so not perceived as painful. Counter irritation: rub injury site to soothe pain TENS: transcutaneous electrical nerve stimulation -> passage of small current with + A fibres

Transmission between nociceptive afferents (A & C) and dorsal horn interneurons is mediated by: Glutamate acts on AMPA receptors -> fast EPSP o Metabotrophic: mGluRs o Ionotrophic: AMPA, NMDA (Mg 2+ block) Neuropeptides i.e. substance P acts via NK1 receptor to elicit slow EPSP allowing for temporal summation & glutamate response Intrathecal injection of substance P conjugated to saporin destroys NK1 expressing neurones in the dorsal horn

 Rats treated with this

neuropathic pain

signs of hyperalgesia in inflammatory &

 But substance P KO & NK1 KO mice show little reduction in hyperalgesia

and NK1 antagonists were disappointing

 Concluded that NK1 is important for hype ralgesia but not mediated by SP
on NK1 but glutamate acting on cells (substance P just heightens it) Two types of pain: y y Nociceptive pain Clinical pain o Inflammatory o Neuropathic

Pain is useful because it warns us of actual or potential tissue damage but if pain systems become too sensitive it will cause us pain that provides us of NO benefit. HOW PAIN SYSTEMS CHANGE:

 Allodynia: when thresholds are lowered so innocuous stimuli produce pain  Hyperalgesia: noxious stimuli produce exaggerated/prolonged pain
Caused by peripheral and central sensitisation Peripheral sensitisation = reduction in the threshold and increase in responsiveness of peripheral ends of nociceptors due to actions of inflammatory chemicals o Primary hyperalgesia region of damage hyperse nsitive to heat and thermal stimuli Central sensitisation = increase in excitability of neurones within the CNS caused by a burst of activity in the nociceptors which alters strength of synaptic connections between nociceptors and spinal cord neurones o Allodynia

Secondary hyperalgesia region outside injury site becomes hypersensitive to mechanical stimuli

o Spontaneous pain Inflammatory pain NEUROTROPHIC FACTORS regulate the long-term survival, growth or differentiated function of discrete populations of nerve cells i.e. NGF (nerve growth factor) NGF- highly basic protein made of 120 amino acids exist as homodimers (2 identical polypeptid e chains) important in the development of the nervous system Binds low affinity p75 receptors and high affinity tyrosine kinase receptors (trk).

p75 preferred receptor for proNGF and modulates the activity of the trk receptors.

TrkA NGF (trkB BDNF/neurotrophin 4 & 5; trkC neurotrophin 3 [but in vitro neurotrophin 3 can activate all of them)

NGF binds -> receptor dimerization ->autophosphorylation of receptor -> tyrosine kinase domain activated -> phosphorylates SH2 receptors on other proteins inc:

 

PI3-K MAP-K (via ras)

trkA principally expressed on peptidergic, small diameter DRG cells ASSOCIATION BETWEEN NGF AND INFLAMMATORY PAIN:

Systemic NGF administration induces thermal hyperalgesia in rodents within 30 mins and thermal & mechanical hype ralgesia after a few hours o Subcutaneous NGF injections produce thermal and mechanical hyperalgesia at injection site

  

IV injections of NGF in humans produce widespread aching pains in deep tissues and hyperalgesia at injection site Elevated NGF in human inflammatory states i.e. bladder of cystitis patients, synovial fluid of arthritis patients Intraplantar injection of carrageenan produces acute inflammatory reaction and thermal hyperalgesia is prevented if patient given trkA -IgG too

Anti-NGF antibodies & NGF antagonists also reduce hyperalgesia

NGF produces sensitization of nociceptors: Short term changes: DIRECTLY = binds to trkA receptors on nociceptors -> phosphorylates PLC- -> PLC- converts PIP2 to DAG -> PIP2 mobilises Ca2+ stores from endoplasmic reticulum via IP3 -> DAG activates PKC -> PKC phosphorylates receptors and ion channels inc. TRPV1, Nav 1.8 -> increase Ca and Na -> depolarisation INDIRECTLY = mast cell proliferation & degranulation via trkA -> histamine, serotonin & numerous cytokines

Limiting amounts in skin -> cytokines produced during inflammation cause proliferation of fibroblasts and keratinocytes -> release more NGF -> activate nociceptors

May also target eosinophils, B - and T- proliferation

Activate sympathetic efferents -> release eicosanoids -> activate nociceptors (but COX inhibitors dont affect NGF induced hyperalgesia)

May activate 5-lipoxygenase which converts arachidonic acid into leukotrienes ->chemoattract neutrophils (neutrophils may be import ant for sensitisation coz animals without them dont show thermal hyperalgesia to NGF)

Long term changes: Alters gene expression too via Ras -MAPK pathway which promotes CREB which induces gene expression inc. TRPV1, ASICs, P2X3 and Nav1.8 -> retrograde transport to DRG (cell body) MAPK (and other erk fragments) translocate to nucleus where they phosphorylate and activate various transcription factors which lead to gene expression -> increased numbers of Inflammation can also create a humoral cytokine sig nal which produces IL -1B which recruits and activates microglia (neuropathic pain) Can lead to central sensitisation -> activation and sensitisation of primary afferents lead to enhanced spinal cord input which may trigger central changes/increased NT or neuromodulator release from nociceptive terminals in DH -> wind up etc. Spinal cord processing of pain Causes of central sensitisation: 1) Wind up a. Homosynaptic (only activated synapse shows change) b. Increase in DH neurone output over the course of a train of inputs c. Corelease of neuromodulators i.e. substance P (acts via NK1 receptor) and CGRP which activates metabotrophic receptors -

>temporal summation -> Mg2+ block removed from NMDA receptors -> voltage gated Ca2+ currents d. Activity dependent; stops after stimulus stops 2) Classical heterosynaptic central sensitisation a. Heterosynaptic b. Activity dependent; outlasts stimulus by 10s of minutes c. Reduction of threshold due to AB recruitment/DH neurones increase number of Aps to suprathreshold stimuli/expand receptive fields (secondary hyperalgesia) d. Nociceptors release glutamate which bind AMPA, NMDA and mGlus& SP which binds NK1 and BDNF which binds trkB -> increase in Ca2+ -> activates serine/threonine protein kinases i.e. PKA, PKC, CAMKII &Src -> phosphorylate NMDA (NR1, NR2A & NR2B subunits) & AMPA receptors increasing sensitivity (increase open time, remove Mg2+) -> PKC & PKA + ERK which phosphorylates potassium channels -> PKC also inserts AMPA receptors into membrane i. MK-801 , NMDA receptor antagonist, reduces pain hypersensitivity but not always i.e. dont contribute to mechanical hypersensitivity following bee venom inflammation, Sorkin et al. ii. Not sure phosphorylation of which subunit is important -> studies have shown that NR2B is most important & blocking its phosphorylation correlates with reduced hyperalgesia BUT contradictory findings e. Noxious stimuli and inflammation + expression of c -Fos& COX-2 (early genes) and prodynorphin, NK1 and TrkB (late genes) via ERK dependent activation of CREB in spinal cord neurones f. Primary afferents are also affected -> increased levels of substance P and BDNF/changes in expression of hundreds of genes that alter their excitability & transmission properties/phenotypic switch in some DRG neurones so s ome begin to express substance P and BDNF so some non-nociceptor afferents can induce central sensitisation =>allodynia 3) Long term potentiation a. Homosynaptic enhancement of EPSPs

b. Brief duration, persistent, high-frequency nociceptor stimulation i. Nociceptors do not normally fire at high frequencies so LTP probably constrained to intense stimuli c. NMDA-receptor induced Ca2+ influx -> activation of CAMKII -> phosphorylation of AMPA receptors & trafficking of AMPA receptors to synapse Opioids and pain control Opioid any substance whose actions are reversed by naloxone y Includes a number of opiates derived from opium (dried sap from seed capsule of poppy) o Morphine (potent) o Codeine (weak) o Thebaine (opioid precursor) Act on (at least) 3 subtypes of opioid rece ptor:

 Mu () brain (cortex, thalamus, PAG), spinal cord (substantiagelatinosa)

o 1 - supraspinal analgesia/ 2 respiratory depression, miosis, euphoria, gastrointestinal motility, physical dependence

 Delta ( ) brain (hypothalamus, PAG), spinal cord (substantiagelatinosa)

o Spinal analgesia, sedation, miosis, inhibits ADH release

 Kappa ( ) brain (pons, amygdala, olfactory bulbs)

Have been cloned and their cDNA sequences deduced -> allowed us to determine their structure and function. The opioid peptides 3 families derived from 3 pro-hormones: -endrophins from POMC - & Dynorphins from pro-dynorphin receptors receptor receptor

Enkephalins from pro-enkephalin

All share the same N-terminal sequence: Try-Gly-Gly-Phe-Met/Leu All belong to G protein -coupled receptor superfamily -> 7 hydrophobic transmembrane domains interconnected by short loops & have an extracellular N-terminus & intracellular C-terminus.

 Mu, delta & kappa = highly homologous (tran smembrane domains &
intracellular loops best conversed) o Transmembrane domain form an opioid binding pocket that is similar across all receptors

 Extracellular loops, N- or C- termini differ greatly

o Deduced which loops were important for binding of ligand s using loss-of-function & binding rescue experiments

  

E1 & E3 = mu selective compounds (DAMGO) E3 = delta selective compounds (DPDPE) E2 = kappa selective compounds

All coupled to G i/G0 proteins (confirmed using Pertussis toxin) (-) AC -> cytoplasmic events o Confirmed using forskolin -stimulated cAMP accumulation (+) MAPK -> transcriptional activity of cells (-) insulin receptor signalling cascades Activation of all 3 types inhibits Ca 2+ entry via voltage gated Ca 2+ channels Activation of / receptors activate inwardly rectifying K + channels Predominantly found on presynaptic nerve terminals & inhibit NT release but some found postsynaptically& hyperpolarise cell neuronal excitability.

Spinal analgesia: act to inhibit release of glu tamate & substance P from primary afferents/hyperpolarise & decrease excitability of projection neurones Supraspinal analgesia: noxious stimuli (+) amygdala, parabrachial area & central grey -> emotional aspects of pain/(+) thalamus -> sensory aspects o Exogenous injection of morphine/glutamate/electrical stimulation into PAG & RVM ->antinociception

Two types of cell in RVM: y ON cells pronociceptive ( firing with nociceptive spinal reflexes i.e. tail flick/paw withdrawal) o Express opioid receptors . .. when opioids from PAG present y OFF cells antinociceptive

o Dont express opioid receptors; opioids bind GABAergic neurones hyperpolarising them dis inhibiting these so 5-HT can be released y Neutral cells dont know what these do opioids (+)periaqueductal grey & nucleus reticularisparagiganticellularis -> (+) nucleus raphe magnus -> (+) serotonin/encephalin pathways (+ noradrenaline from LC) -> decrease transmission of nociceptive information through DH ANTIOPIOID SYSTEM up-regulation of CCK mRNA following nerve injury in DRG -> mobilises Ca 2+ from intracellular stores/decreases the availability of enkephalins/activates ON cells to induce thermal hyperalgesia Weak CCK antagonist (proglumide) acts on CCK receptors & enhances opioid analgesia o Unfortunately, CCK 2 selective antagonist didnt augment analgesic effect of morphine in patients with chronic neuropathic pain Neuropathic pain: smaller & spatially restricted loss of opioid receptors/up regulated Ca 2+ channel activity/increased excitation of spinal neurones due to activation of NMDA receptors i.e. windup

Other subtypes have been postulated i.e. 1& 2 Recently, an opioid -receptor like orphan receptor has been cloned ORL1 o Believed to bind orphanin FQ (nociceptin) [17 aa peptide] but physiology of system still unknown

Possibly involved in central modulation of pain (NOT associated with respiratory depression)

Opioids used clinically primarily activate receptors Morphine Diamorphine (heroin) more lipophilic and more potent than morphine Codeine weak opioid (mild pain) Pethidine labour & minor surgery o Plasma half-life of morphine is 3-6 hrs& longer in neonates since they lack appropriate liver enzymes/also risk of neonatal respiratory depression

Pethidine doesnt rely on liver metabolism so safer

Methadone long acting, once daily dosing, used to treat addicts Buprenorphine very lipophilic, partial agonist, used to treat addicts Fentanyl very lipophilic, short-acting, used as part of pre -medication for surgery (patches used in cancer patients) Analgesia used to treat mild-severe pain; less effective against neuropathic pain Euphoria state of optimism, cheerfulness and well -being Respiratory depression medulla becomes less sensitive to change in pCO 2 -> cause death in overdose (naloxone used to treat overdose) Nausea and vomiting administered along with an anti -emetic which blocks effect of chemical trigger zone in area postrema Miosis pin point pupils in overdose Inhibition of cough reflex NOT opioid effect; (+) dextromethorpham receptor? Used in cough remedies

Severe constipation due to high level of receptors on enteric nerves kaolin & morphine used to treat diarrhoea Contract gall bladder & constrict bilia ry sphincter use with care in pain of gallstones Dependence psychological (take drug for its pleasurable effects)/physical (stop unpleasant side effects) -> opioids produce both Related to development of tolerance caused by adaptive changes i.e. AC levels Most opioids administered orally or by injected/buprenorphine given sublingually/codeine &diamorphine are metabolised to morphine in the body -> morphine metabolised in liver & metabolites excreted in urine Purines and pain control P2 purinoceptors: y y Ionotrophic (P2X) Metabotrophic (P2Y)

Bind ATP P2X receptors ligand gated ion channels cation selective channels (Ca 2+ mostly but also Na +& K+) y Molecular cloning identified 7 genes encoding P2X subunits o All possess 2 transmembrane regions & have intracellular N and C termini & a long extracellular loop between the transmembrane regions


y

1/3rd of aa in extracellular loop are conserved in ~ 6 subunits -> involved in ATP binding

Functional P2X receptor channel formed by a number of P2X subunits (like most other ionotrophic receptors) -> possibly 3 or 4 o Can be homomers/heteromers

P2X4 1. Activated microglia* after nerve injury increase expression of P2X receptors

a. Activation of microglia is TLR2/4 dependent (LPS from pathogens/endogenous ligands bind & activate b. CCL2 from damaged nerves can also stimulate CCR2 receptors on microglia 2. ATP released from primary afferents/astrocytes bind to P2X4 receptors 3. intracellular Ca2+ & activation of p38MAPK (affects transcriptional activity) &proinflammatorychemokines (CatS) and cytokines (IL-1) which acts directly on neurones a. Release of bioactive diffusible factors also caspas e 1 and its accessory protein ASC dependent b. CatS cleaves soluble transmembranefractalkine -> this interacts with CX3CR1 receptors on microglia & (+) further release of diffusible factors i. Cytokines &chemokines attract more spinal microglia & astrocytes 5. BDNF down-regulates KCC2 channels, which renders GABAergic currents from inhibitory interneurons depolarising 6. May interact with excitatory synapses of neighbouring DH neurones & excitability *Activated microglia show amoeboid morphology, more phagocytic ac tivity & increased immune molecule expression (when quiescent exhibit ramified processes which are highly motile & express low levels of immune molecules) P2X3 can form homo-oligomeric channels or assemble with P2X2 subunits into hetero-oligomers ATP causes pain applied ATP by ionophoresis into the skin of human fore -arms -> elicited mild sensation of pain but pain was enhanced by other inflammatory mediators i.e. capsaicin/UV light/nocifensive behaviour such as hindpaw licking & lifting following sub plantar administration of ATP like in humans, increased nocifensive behaviour when coapplied with prostaglandin E2/formalin/carrageenan P2X3 receptors are found on non -peptidergic sensory nerurones clear excitatory effects of ATP & meATP from in vitro recordings of skin nerves 4. Release of bioactive diffusible factors i.e. BDNF

from rats -> increased in preparations when skin had previously been inflamed with carrageenan y In P2X3 KOs, non-peptidergic C-fibres are intact meATP:

Two kinds of response to ATP &

Rapidly rising inward current that desensit ises during maintained applications -> homo-oligomeric Slowly rising inward currents that declines little if at all during repeated applications -> hetero-oligomeric(likely to play a physiological role) Where does endogenous ATP come from? DAMAGED TISSUE y Primary afferent nerves o Sympathetic nerves copackaged with noradrenaline i.e. in vas deferens/arteriolar smooth muscle y Endothelial/epithelial cells o Release of ATP from bladder epithelia in response to distention -> acts on P2X3 receptors on primary afferents beneath urothelium -> initiates voiding (start urination)


y

P2X3 KO mice must be distended to a larger volume before voiding reflex initiated

Lysed cells rheumatoid joints have more ATP than normal

Chronic inflammation (complete Freunds adjuvant administered to hindpaw) -> expression of P2X3 protein expression in dorsal root ganglia & larger than normal ATP-induced currents. Evidence that it plays a role in pain: y Antagonists o Sumarin reduces pain in man but it has actions at so many different receptors so findings cannot be attributed to P2X o TNP-ATP reduces nocifensive behaviour elicited by intraplantar injection of formalin/intra -abdominal injection of acetic acid almost as potent as morphine

Blocks P2X1 & P2X3 receptors but using P2X1 receptor blocker determined antagonism of P2X3 caused effects

o A-317491: P2X3 antagonist produced analgesia following complete Freunds adjuvant (inflammatory)/chronic constriction injury (neuropathic) in rat species o PPADS y P2X3 subunit knock down with antisense oligonucleotides expression of P2X3 in DRG -> marked reduction in chronic (but not acute) inflammation induced thermal & mechanical hyperalgesia& spinal nerve ligation induced mechanical allodynia y P2X3 gene KOs o Homozygotes no detectable P2X3 protein in afferents, dorsal root &nodose ganglia -> no currents elicited by ATP analogues

  

Modest reduction in hindpaw licking & lifting after intraplantar formalin Paradoxical potentiation of thermal hyperalgesia following complete Freunds adjuvant Impairment in ability to sense bladder filling

Mechanisms of neuropathic pain Neuropathic pain: pain initiated or caused by a primary lesion or dysfunction in the nervous system; can be: y y y Peripheral Central Mixed

Causes of neuropathic pain: 1) Infection i.e. HIV, herpes simplex virus 2) Metabolic i.e. diabetic neuropathy 3) Neurotoxicity i.e. chemotherapy (vincristine) 4) Traumatic i.e. surgical damage 5) Central i.e. SCI Symptoms: paradoxic because cutting axons should dea den sensation but there is also evidence of:

 Abnormal sensations which occur spontaneously o Burning/coldness o Cramping o Pins and needles sensation o Itching Pain hypersensitivity o Allodynia pain towards innocuous stimuli

Mechanical (tactile)

 Static pain in response to light touch/pressure  Dynamic pain in response to brushing

o (Hyperalgesia stimuli produce exaggerated/prolonged response)

 

Primary (damaged tissues) -> heat Secondary (surrounding undamaged tissues) -> mechanical

Can be continuous/episodic (paroxysmal)* *Likened to electrical shock Animal models of neuropathic pain: one a portion of the afferents going to the foot arelesioned

 Spinal nerve ligation one or more spinal nerves going to foot are ligated
and cut

 Partial sciatic ligation portion of sciatic nerve is tightly ligated  Chronic constriction injury placement of 4 loos chromic -gut ligatures on
sciatic nerve -> immune response leads to nerve swelling and constriction

 Spared nerve injury common peroneal&tibial nerves cut but sural nerve
left intact

 Streptozotocin model of diabetes associated with development of

neuropathy Pain hypersensitivity easy to measure in animals (i.e. Von Frey Test of Mechanical Threshold, Radiant Heat Test of Thermal Sensitivity) bu t abnormal pain sensations difficult [autotomy (self -amputating limbs), cellular markers of increased neuronal activity i.e. c -Fos, fMRI/PET imaging)

 Animal models in some cases have been inconsistent with human models ->
diabetic neuropathy infrequent in humans but robust in rats; NK1 works in animals not humans

 Reflex measures of pain i.e. paw -withdrawal threshold method tests

efferents NOT afferents Possible mechanisms Spontaneous neural activity & ectopic sensitivity to mechanical stimuli at the site of nerve injury Expression of different molecules in the DRG of injured nerve is up/down regulated reflecting loss of trophic support from periphery & spontaneous neural activity in the DRG Distal part of injured nerve undergoes Wallerian degeneration exposing surviving nerve fibres to cytokines & growth factors Partial denervation of peripheral tissues leads to an excess of trophic factors tissue leading to peripheral sensitisation Expression of different molecules in the DRG of uninjured nerves up/down regulated reflecting enhanced trophic support Sensitisation of postsynaptic DH neurones Activated microglial cells contribute to development of central sensitisation Changes in descending modulation of DH neurones Injured afferent hypothesis y Spontaneous firing in some afferents & abnormal responsiveness to thermal, mechanical & chemical stimuli in others o Neuroma endbulbs proximal end of axon seals off and forms terminal swelling o Regenerating/collateral sprouts fine processes that grow from endbulb and endeavour to reform connections with target tissues

Without growth guidance from Schwann cells form entangled mess which is painful (dont know why genetics? Location?)

o Cell soma in DRG o Patches of demyelination y However, studies found that only A-fibres show ectopia not C-fibres o Problem because C -fibres were thought to induce central sensitisation but A-fibres undergo phenotypic switch & express substance P after nerve injury

y y

Evidence suggests that local anaesthetic blockade of injured ne rves relieves pain Reversed neuropathic behaviours in animal models treated with anaesthetics/TTX

Intact nociceptor hypothesis y y y Average discharge frequency low but incidence of spontaneously firing nociceptors is high Become sensitised particularly to n oradrenaline & TNF Increased responsiveness to heat -> TRPV1

Ectopic neurones -> distort & amplify other Aps with afterdischarges (EADs) and extra-spike formation Growth factors y y Nerve injury induces changes in trophic factors in the tissue of deprivation , Schwann cells without an axon anymore, DRG & dorsal horn Increase in NGF -> binds TrkA -> transport to DRG -> affects factors such as BDNF o Regulates TRPV1 expression o Abnormal expression of transduction molecules o Threshold for activation of transduction decreases i.e. sodium channels -> Nav1.3 is unregulated in DRG of injured axons

 Sodium channels accumulate in membrane at sites of nerve

injury y Undertrophed neurones which have lost connectivity -> without GDNF become ectopic (treatment with GDNF r everses neuropathic pain behaviour) y Overtrophed neurones -> too much NGF?

Central sensitisation y Homosynaptic (windup)/heterosynaptic mechanisms -> increased release of excitatory NTs/enhanced synaptic efficacy o Presynaptic changes

Released of glutamate inhibited by -opiod receptors, GABA B and adenosine receptors -> down-regulated -> glutamate release

 

Up-regulation of voltage-gated Ca 2+ channels -> glutamate release A fibres undergo phenotypic switch & express substance P

 Synapse in substantiagelatin osa& form synapses with

projection neurones o Postsynaptic changes

    

AMPA receptors on cell surface (and NMDA in diabetic neuropathy) substance P and other peptide release Necrosis of inhibitory interneurons LTD evoked by activation of NMDA receptors on GABAergic neurones Down-regulation of KCC2 symporter so opening Cl - by GABA induces depolarisation -> KCC2 KOs show hyperalgesia

o Interneurone changes

o Changes in descending modulation

RVM modulates inputs from cortical, thalamic & periaqueductal areas -> decides whether to excite or inhibit projection neurones

Ablation of -opiod receptor expressing cells -> decreased inhibition

o Immune/microglial mechanisms

Nerve damage ->denervated Schwann cells screte LIP & MCP-1 -> macrophage infiltration, T-cell activation &proinflammatory cytokines

 MCP-1 -> receptor for CCR2 (KOs show no

hyperalgesia)

 IL-1 -> NGF, TNF 

Central microglia

Eph B receptors and pain modulation at spinal cord level Understanding the tyrosine kinase family of Eph receptors and their ligand, the ephrins

and B subclasses y Bind transmembrane proteins called ephrins

EphB-ephrin B2 forms a ring-like tetrameric structure with each ligand interacting with 2 receptors and each receptor interacting with 2 ligands.

Unde

nding their role in development

Guide migrating cells and neuronal growth cones towards their targets, modify cytoskeleton organisation and cell adhesion. Knowing something about their mechanism of action EphB receptor auto-inhibited via interaction with its own uxtamembrane domain. On activation phosphorylation of uxtamembrane tyrosine residues removes inhibition. Why EphB and ephrins B are related to sensory systems mmunohistochemistry has shown the presence of EphB receptors in the
  

superficial laminae of the dorsal horn and ephrin B2 in the dorsal root ganglion.

Largest amily

tyrosine inase receptors

4 members ivided into

  

Ephrin B2 agonists injected into the lumbar spinal cord resulted in a short latency & prolonged behavioural thermal hyperalgesia, indicated by a 50% reduction in withdrawal latencies to noxious heating of the hindpaws in the plantar test o Role in inflammatory pain?

y y

This response was blocked by the NMDA receptor antagonist MK801 Treatment with EphB1 receptor antibodies reduced thermal hyperalgesia and mechanical allodynia following carrageenan intraplantar injection in adult rats & c-Fos expression was reduced in DH o Injecting EphB1 antibodies before formalin reduced pain related behaviour

Reduction in c-Fos expression in EphB1 KOs after carrageenan injection

Understanding the role of EphB in modulating pain processing at spinal cord le el interacting with NMDA glutamate receptors Ephrin B2 -> Activated EphB1 receptor -> phosphorylation of NR2 subunit of NMDA receptor by src -> amplification of NMDA activity Evidence: y y y Administering Ephrin B2 increases src EphB receptor contains YEPD motif which binds to src Phosphorylation of NMDA-R by EphB2-Fc prevented by prior injection of PP2 (src blocker)


GLIAL NEUROBIOLOGY Two types of brain cell y y Neurones (excitable) Glia (non-excitable) most populous; ~90%

Types of glia: y CNS o Microglia (non -neuronal -> originate from macrophages)

Ameboid found in fountains of microglia (corpus callosum) so free movement -> prevalent in perinatal brain

(development when there are lots of extracellular debris & apoptotic cells)

Ramified mature, resting -> found in strategic locations throughout brain (regular mosaic pattern)

o Macroglia

  
y PNS

Astrocytes Ependymal cells Oligodendrocytes

o Schwann cells ?Synantocytes y Express the NG2 chondroitin sulphate proteoglycan (CSPG) -> considered to be oligodendrocyte precursor cells that persist in the adult CNS to generate oligodendrocytes throughout life o Not in same place as oligodendrocytes or express markers y y Stellate cells with large processes that form contacts with neurones at synapses and nodes of Ranvier Neuronal activity (glutamate & ATP) act on receptors & evoke intracellular Ca -> proliferate & change phenotype (becom e oligodendrocyte/neurone) -> physiological function? Maybe specialised to monitor signals from neurones and glia Glial cell development: Neuroepithelium (neural progenitors) forming neural tube -> radial glial cells -> generate majority of neurones & macroglia in CNS (neurogenesis) from precursors (i.e. SC precursors) o RGCs also form scaffold along which they can migrate from ventricular zone to their home o RGCs eventually become astrocytes

Small proportion become stem astrocytes for adult neurogenesis

Astrocytes (star-like)

 Most populous glial cells in CNS

 ALL express intermediate filament protein: glial fibrillary acidic protein

(GFAP) or vimentin which forms cytoskeleton [levels vary btw cells] o High tensile strength -> gives CNS its structure Types of astrocyte: y True astrocytes (with classical stellate appearance) o Fibrous

   
y

White matter Elaborate and complex processes Grey matter Less elaborate but longer processes

o Protoplasmic

Radial glia (disappear following maturation & turn into true atsr ocytes; some persist i.e. Muller cells in retina, Bergmann cells which support Purkinje cells in cerebellum)

Specialised astrocytes

Functions of astrocytes 1) Structure a. Each protoplasmic astrocyte occupies a well -defined territory i. Overlap between astrocyte territories doesnt exceed 5% b. Using these territories, astrocytes divide grey matter into domains i. Neurones, synaptic terminals & blood vessels are integrated by distal processes of astrocytes 2) Blood brain barrier a. Astroglialendfeet enwrap capillary wall & release regulatory factors i.e. GDNF & TGF which induce formation of tight junctions between endothelial cells i. Endothelial cells communicate reciprocally with astroglia via LIF b. Endfeet endowed with glucose transporters, potassiu m channels and water channels -> absorbs these and sends it to neurones in its domain c. Regulate cerebral blood flow

i. Increased blood flow: Glutamate release at synapses -> activates Ca 2+ influx -> propagates to end-foot ->arachidonic acid release -> converted to PGs by COX -> vasodilation ii. Decreased blood flow: Less glutamate release at synapses -> less Ca 2+ -> propagates to end-foot -> AA release -> converted to 2-HETE by P450 -> vasoconstriction 3) Neurotransmitter uptake a. Remove glutamate released by neurone s thru EAAT1 & EAAT2 -> convert to glutamine -> glutamine taken up by presynaptic terminal -> recycled to glutamate i. Terminates signal, recycles, protects against neurotoxicity 4) Metabolism a. Astrocytes taken up glucose via GLUT1 -> convert into pyruvate -> convert into lactate (using lactate dehydrogenase) -> glutamate released during neuronal activity + release of lactate -> lactate taken up by neurone (via monocarboxylase transporters) [ACSTROCYTE-NEURONE LACTATE SHUTTLE] 5) Potassium homeostasis a. Potassium released into extracellular space during neuronal activity (repolarisation) -> astrocytes taken up excess K+ thru K irchannels and redistribute the K+ thru the astroglial syncytium via gap junctions (Cx 43, 30 & 26) SPATIAL POSTASSIUM BUFFERING i. Possible because astrocytes express high density of voltage gated potassium channels & have strongly ve RMPs 6) Neuron-glial signalling a. Astrocytes express NT receptors (glutamate, ATP) -> when activated produces IP 3 which + ER to release Ca 2+ -> Ca 2+ transmitted via diffusion thru gap junctions (INTERCELLULAR PROPAGATION) -> release of gliotransmitters which affect neurone activity i. Can occur at neurone -glial/tripartite synapses (pre-, postsynaptic neurone & astrocyte; gliotransmitters can affect metabotrophic recept ors on pre- and ionotrophic on post) Oligodendrocytes, Schwann cells and myelination

Oligodendrocytes y 4 types: o Type 1/2 myelinate small diameter axons; 1:30 o Type 3 myelinate large diameter axons; 1:3-5 o Type 4 myelinate large diameter axons; 1:1 Schwann cells y 3 types: o Myelinating ensheath axons > 1 m diameter; 1:1 o Non-myelinating ensheath multiple axons < 1 m diameter; physical support & separation, ion homeostasis, express cell adhesion molecules; immature -> can adopt myelinating phenoty pe o Perisynaptic ensheath terminal axon branches & synaptic boutons; covered with basal lamina which fuses with muscle fibre; essential for synapse function y Unlike oligodendrocytes, SCs form basal lamina which is continuous tube along axon and bridges the nodes o SC microvilli fill the nodal gap & are important for potassium regulation at nodes


Myelin sheath Made of: y y

In PNS microvilli are formed by astrocytes & NG2 -glia

70% lipid insulating properties 30% proteins fuse & stabilise lamellae & mediate membrane-membrane interactions (MAG mediates interactions in both) o Major proteins in CNS:

 

MBP fuse cytoplasmic interface forming major dense line PLP fuse extracellular interface forming intraperiod line

o Major proteins in PNS: Charcot -Marie-Tooth neuropathy (dysfunctional proteins)

  

P0 mediates adhesion of lamellae PMP22 myelin synthesis & assembly; also joins Schwann cell to basal lamina Cx32 gap junctions for ion homeostasis

De elopment


Schwann cells: neural progenitors (if exposed to delta/notch, NRG -1 & transcription factor SOX10) -> SC precursor -> immature SC (if exposed to NRG-1 & transcription factor Krox-20) ->myelinating/non -myelinating SC

Oligodendrocytes: develop in ventricular zones under influence of transcription factors and signalling molecules -> migrate to final sites in CNS & undergo local proliferation & differentiation in response to growth factors from astrocytes & interactions with axons

Axon-glial interactions in the control of myelination 1) Myelinating cells recognise which axons to myelinate when SC integrins/periaxin binds L1 & when OL neurofascin binds L1 a. NCAM also plays a role b. Jagged/Notch prevents myelination by OLs 2) SCs & OLs organise sodium channel cluster ing at nodes of Ranvier Multiple sclerosis BBB normally prevents most lymphocytes & phagocytes entering CNS. This coupled with a poor lymphatic drainage system & low expression of MHC means antigen presentation is poor. In inflamed state, BBB break dow n -> induces MHC expression on microglia & endothelial cells -> interact with T-helper cells y Episodes of neurological dysfunction secondary to inflammatory lesions within CNS confined to small areas of demyelination o Lesions often resolve with remyelinati on& clinical recovery but with time, permanent loss of myelin occurs with secondary axonal loss & fixed disabilities

Oligodendrocyte precursor cells proliferate &remyelinate the axons but ultimately fail due to lack of growth factors & too much inhibition

Events that occur: 1) T-cells activated against myelin components

2) T-cells attack myelin & activate microglia 3) Activated microglia release cytokines, activate astrocytes & attack oligodendrocytes& neurones

Alzheimers disease Dementia progressive decline of cognitive function usually affecting cortex as a whole but sometimes patchily; impairment of intellect, memory & personality without disturbance of consciousness Alzheimers disease CLINICAL FEATURES AND DIAGNOSIS y y Commonest dementia (accounts for ~65% of dementia in any age group) Cardinal clinical features: o Amnesia (memory loss) inability to learn, retain & process new information o Aphasia (decline in language) difficulty in naming and in understanding what is being said, simplification of language, descriptive power declines -> complete loss of communication o Apraxia impaired ability to carry out skilled motor activities i.e. eating, dressing, drawing, waving goodbye despite intact sensory & motor systems o Agnosia failure to recognise object i.e. prosopagnosia (faces), places, own body parts o Mood disorders, delusions, hallucinations, misidentifications, behavioural changes (a gitation, aggression, wandering) y Differential diagnosis: delirium, pseudodementia, vascular dementia, dementia with Lewy bodies, fronto -temporal demntia (difficult to recognise each type of dementia) o Also difficult to define landmarks that separate demen tia from mild decline in cognitive function associated with ageing y Diagnosis based on: o History (particularly collateral history from informant i.e. carer) o Mental State Examination

o Physical examination esp. cardiovascular risk factors & neurological problems o Investigations i.e. blood, urine, imaging

AETIOLOGY y Early-onset AD (familial emerges before 65 yo) AUTOSOMAL DOMINANT (1/2 the offspring of affected parents will get disease) o Amyloid precursor protein (chr. 21)

Increased incidence in Downs syndrome sufferers by 40 yo

o Presenilin 1 (chr. 14) o Presenilin 2 (chr. 1) y Late-onset AD (sporadic) o ApoE4 (chr. 19 -> encodes glycoprotein, apolipoprotein E; involved in cholesterol transport & metabolism; 3 alleles ApoE4: high risk)

  

No copies lower risk than population 1 copy 4 fold increase in risk 2 copies 8 fold increase in risk

o Epidemiology risk factors: age, head trauma, cholesterol, CVD PATHOPHYSIOLOGY 1) Gross cerebral atrophy

2) Senile plaques

Spherical lesions measuring up to 100 m o Fully developed stage (neuritic plaque) central core of amyloid surrounded by dystrophic neuronal processes, reactive astrocytes & microglia


y y

Diffuse plaques also present

Amyloid precursor protein transmembrane protein that projects into ECF from all nerve cells Can be hydrolysed at 3 different sites by formed (A
40

-, - or -secretase

o When hydrolysed by -secretase, nontoxic peptide products not prone to aggregation) - or -secretase, polypeptides with 40-42 o When hydrolysed by

aas are produced that are toxic

 

Most toxic = A

42

Form extracellular aggregates (together with altered nerve fibres and reactive glial cells) -> stick to AMPA receptors & Ca 2+ channels -> Ca2+ influx -> apoptosis y They can bind to surface receptors i.e. prion proteins (also linked with CJD) on neurones and change synaptic communication

  

Initiate inflammatory response & inhibit neurotrophic factor release -> intracellular tangles Also builds up in the mitochondria o f cells and inhibit certain enzymes disrupting glucose metabolism N-APP (fragment of APP) may trigger cells to self-destruct by binding onto death receptor 6

Astrocytes detect -amyloid released by affected neurones and withdrawtheir process from affected & neighbouring neurones -> these neurones die without support -> the -amyloid released accumulates in astrocyte -> astrocyte death -> debris attracts microglia -> plaque formation 3) Neurofibrillary tangles y y Found in neurones, senile plaques & dendrites Hyperphosphorylation of tau protein (microtubule -associated protein) by active GSK-3 which pairs with other threads to form paired helical filaments

y y

Microtubules disintegrate -> transport system collapses Interfere with cellular functions by displacing organelles

4) Neurone and synapse loss y Starts in the transentorhinal cortex (hippocampus &neocortex)

5) Neurotransmitter changes y Acetylcholine o Loss of cholinergic neurones in the basal forebrain, decreased acetylcholine levels and a decrease in choline acetyltransferase (CHAT) which synthesises ACh o Animal models show that ACh plays a crucial role in information processing & memory o Senile plaques may affect cholinergic synapses --> in vitro studies show ACh release


o

Reductions in APP in depr essed patients CSF who are taking drugs with anti-cholinergic side effects

ACh -> (-) PKC -> increased activity of GSK-3 >hyperphosphorylation of tau

Glutamate o Inactivation of EAAT1, EAAT2 & glutamine synthetase -> glutamate in synaptic cleft o VGlut1 which pumps glutamate into vesicles coupled exocytosis (synaptophysin?) o Vesicular glutamate released into cleft by Ca 2+ dependent stimulus

Interesting to note that tetrahydro -9 aminoacridine improves cognitive performance in some AD patients & reduces synaptic Ca 2+ coupling & glutamate release

glutamate -->excitotoxicity which kills cells by necrosis & apoptosis; activation of mGlu2 can exacerbate problem

Activation of NMDA receptors causes dissociation of protein phosphatase-2A --> tau phosphorylation

o Not beneficial for LTP because tonic rather than phasic stimulation of NMDARs --> background noise which impedes detection of relevant signals (partial depolarisation -> background Ca 2+ influx)

Decreased numbers of pyramidal and stellate cells that stain for glutamate and glutaminasein hippocampus -> neurones that are present have disorganised and shortened dendrites

AMPAkines positive modulators of glutamate action at AMPA receptors/increase production of neurotrophins i.e. BDNF & NGF in brain areas involved in memory using slices of brain tissue from aged rats & mice

Memantine moderate affinity, non -competitive NMDA receptor antagonist MK801 high affinity, competitive NMDA receptor antagonist

Serotonin o Reduction of 5-HT in post mortem AD brains -> 5-HT reuptake sites in temporal cortex /Raphe nucleus preferential site for NF formation/ 5-HT2A receptor o Frequent co-existence of depression?

Spinal cord injury and repair Describe the gross anatomy of the spinal cord EXTERNAL FEATURES y y y Provides sensory, motor & autonomic innervation for the trunk and limbs Occupies the spinal canal within the vertebral column ~ cylindrical but diameter varies at different levels o Bears 2 enlargements:

 
y

Cervical provides innervation for the upper limb via the brachial plexus Lumbar innervates the lower limb via the lumbar & sacral plexi

Terminates at vertebral level L1-2 in the adult (conusmedullaris) o Filumterminale [connective tissue] attaches conusmedullaris to first coccygeal vertebra

31 pairs of spinal nerves attach to the spinal cord through dorsal & ventral roots, carrying afferent & efferent fibres respectively o Exit the vertebral canal via intervertebral foramina o Below termination of cord, spinal nerves descend as caudaequina

o Subarachnoid space between pia& arachnoid mater) contains CSF N ERN L FEATURES Dorsal funiculus Dorsal horn Lateral funiculus Lateral horn
" ! 

Dorsal medial sulcus

Ventral funiculus Ventral white commissure y y

Ventral median fissure

#

Central canal continuous with cerebral ventricular system contains CSF) Grey matter nerve cell bodies, dendrites & synapses o Dorsal horn main site of termination of afferents o Lateral horn preganglionic sympathetic fibres o Ventral horn lower motor neurones o Ascending tracts dorsal columns, spinothalamuc tract, spinoreticulothalamic system, spinocerebellar tract o Descending tracts corticospinal tracts, rubrospinal tracts, tectospinal tracts, vestibulospinal tracts, reticulospinal tracts
0 ) ( ' & % $

White matter nerve fibres

Describe the neuropathology of spinal cord injury y y Spinal cord injury refers to any injury that is caused by trauma instead of disease. Depending on where the spinal cord is damaged, the symptoms can vary below the point of injury:
1

widely

generally, weakness/paralysis/spasticity and sensory lossat and

Cord is covered by 3 meninges pia, arachnoid dura mater

Central canal

Ventral horn

o Cervical quadriplegia (+ blood pressure problems, breathing difficulties, abnormal sweating, breathing difficulties, problems maintaining steady body temperature) o Thoracic paraplegia o Lumbarsacral bladder, bowel & sexual dysfunction y All can be described as: o Incomplete partially injured o Complete severely injured Estimated 2.5 million people live with SCI & 130,000 new injuries reported each year significant impact on QoL, life expectancy and economic burden (due to primary care & loss of income). Causes: Motor vehicle accidents Falls Violence Sports injuries Minor injuries inc. whiplash Pathophysiology of SCI 1. Acute a. Haemorrhage occurs -> localised oedema -> loss of microcirculation by thrombosis & vasoconstriction of blood vessels i. Vertebral disruption/oedema compresses spinal cord & exacerbates problem b. Mechanical (injury) & ischaemic insults -> necrosis (particularly in grey matter) c. Injured nerves respond with an injury -induced barrage of APs i. Electrolytic shift -> spinal shock (generalised failure of spinal cord) 2. Secondary a. extracellular glutamate (and other excitatory aas& free -radicals) -> apoptosis forming cyst/cavity which interrupts cord i. Wallerian degeneration (degeneration of axons distal to an injury when it has become separated from cell body)

ii. Myelin disintegrates & fragments but oligodendrocytes survive iii. Reactive gliosis (microglia activated) P2X & glutamate receptors so detect apoptosis Activated astrocytes grow & reproduce -> GFAP, growth factors, inflammatory mediators i.e. IL -1 o Neutrophils which clear debris invade followed by lymphocytes iv. Astrocytes also form glial scar which protects other neurones from damage but blocks regenerati on & reforms BBB (angiogenesis) Synantocytes also help form glial scar & enable oligodendrocytes to survive 3. Chronic a. Cysts/cavities can continue to enlarge (syringomyelia) - FATAL b. Compensatory sprouting (limited) c. Compensatory cortical, brainstem & spinal plasticity (limited) new spinal circuits can bypass the lesion i.e. rubrospinal tract can take over job of corticospinal tract/cortical sensorimotor areas can functionally rearrange d. Little/no neurogenesis but proliferation of ependymal cells can give glial precursors mainly Describe animal models of spinal cord injury Vast majority of research conducted using adult mice (transgenic) & rats (larger) because they are cheap, although some has been done using cats and dogs and increasing work is being done using nonhuman primates. 4 types of SCI

 Cord maceration (morphology seriously distorted) subdural insertion and

inflation of a balloon/clip compression

 Cord laceration (gun shot/knife wounds) surgical incision  Contusion injury (bruising injury) weight drop  Solid core injury none available

Animal model depends on hypothesis neuroprotection (contusion/maceration); pro-regeneration (laceration can distinguish between regenerating axons from spared/collaterals) Problems: No studies showing any improvements after intervening > 1 month post injury (long-standing injuries in humans) Only assess outcome over period of weeks/months -> do these remain stable beyond 2/3 months Bipedal locomotion differs radicall y from quadrupedal locomotion/partially upright locomotion posture, kinematics, physiology, anatomy Differences in responses to SCI between species & members of the same species o Cysts/cavities form in humans but not in mice Animal models indicate that axon regeneration by a small % is sufficient to restore partial function Describe possible mechanisms contributing to loss of function Critically e aluate current treatments (pharmacological, rehabilitati e) REHABILITATIVE 1. Patients are immobilised at scene of injury until they deduce there is no damage to highest portions of the spine 2. Brought to hospital evaluated for SCI using X -ray/CT scan 3. Complications spinal shock -> autonomic dysreflexia/respiratory failure, pulmonary oedema, pneumonia/DVT ( can be recognised early and avoided kept in ICU) 4. Surgery to remove any bone fragments and stabilise the spine (decompress) 5. Treated with corticosteroids (methylprednisolone) to reduce inflammation 6. Rehabilitation a. Support and prevention gives individual sense of control over situation/prevention of pressure sores in bed & wheelchairs
2 2

b. Support and education for individual & caregivers evaluation of limb function to determine what patient can do/selfcare skills/eating, bowel & bladder management/mobili ty/sexual health & function c. Assistive devices i.e. wheelchairs/sliding transfer boards/grab bars d. Patients living environment needs to be modified i.e. ramps/lifts Improved locomotor function seen in mammals with complete/incomplete SCI following exercise. Even enhances ability to walk on a treadmill when body weight is supported spinal circuitry does not become silent but maintains active and functional neuronal properties & can respond to peripheral input from below lesion site can generated oscillating, coordinated motor patterns & considerable plasticity. Functional electrical stimulation (electrophysiological stimulation of spinal cord/peripheral nerves/muscle) can also induce step -like movements in patients with complete SCI. Helps chronic (faster overground walking speeds but patients only used wheelchairs) & recent patients. Combination of treadmill training and spinal cord epidural FES improved quality & quantity of stepping during the training session and resulted in the im mediate improvement in quality of overground walking Epidural + locomotor training = BEST Improved cardiovascular performance, reduced spasticity, bone loss & bladder/bowel dysfunction However, can pose risks i.e. autonomic dysreflexia, fracture, muscula r injury, hyperthermia (atypical responses to exercise) Ichiyama et al. given enough physiological activation (ES + quipazine) afferent input can drive spinal circuits to enable stepping and that repetitive practice allows the isolated lumbosacral circuit s to find new solutions in executing motor & postural problems imposed by SCI stepping more consistent and fewer neurons activated in trained (body weight support system for bipedal stepping) vsnontrained spinal rats

Fong et al. robotic step training and a serotonin agonist, quipazine generated significant recovery of locomotor function in complete spinal cord transected mice that otherwise could not step extent of recovery achieved when these treatments were combined exceeded that obtained when either treatment was applied independently Prostheses: artificial device extension that replaces a missing body parts may supplement/replace biological therapies PHARMACOLOGICAL PNS neurones regenerate long distances by Wallerian dege neration y y y y y Myelinating Schwann cells dedifferentiate and myelin fragments leaving basal lamina intact Macrophages remove debris Schwann cells line up and release growth factors which cause axon to sprout collaterals and regenerate Perisynaptic Schwann cell s reform connections Schwann cells remyelinate axons

CNS neurones grow poorly because: Limited by inhibitory influences of glia & the extracellular environment

 Glia produce factors that inhibit remyelination& axon repair i.e. NOGO
o Orientated perpendicular to the neuraxis so physical barrier

 Axons lose capacity to regenerate due to less GAP 43, RARb  Environment myelin-associated inhibitors, astrocytes, oligodendrocytes,
oligodendrocyte precursors, lack of laminins Chondroitin sulphate proteoglycan

 Astrocytes (+) CSPGs (requires RhoA pathway)  All CSPG inhibitory to neurite outgrowth (projection from axon)
chondroitin-4 proteoglycan was the most inhibitory

 NG2 is a GSPG that is expressed by oligodendrocyte precursor cells in

glial scar -> following injury to CNS, NG2 expressing OPCs are seen around the site of injury within 24 hours of the initial injury and remain

elevated for ~ 8 weeks. In vitro studies show neurite growth inhibition (neurones didnt bind NG2) o Cultured with NG2 & adhesion molecules significantly reduced neurite extension with NG2 than without o Cultures created with striped surfaces NG2 lanes & adhesive molecule lanes neurones stick to lane with no NG2

 Shows CSPGs stop neuronal growth into glial scar  Delivery of chondroitinase ABC (bacterial enzyme) which degrades CSPGs
promotes regeneration of injured CNS axons o Delivery of bacterial proteins carries a risk of nonspecific immune responses & further doses may not be possible if body becomes immune o Human enzymes i.e. hyaluronidases/matrix metalloproteinases might avoid these problems

 Rolls et al. used xyloside to inhibit CSPG formation at different time

points after injury and analysed phenotype acquired by microglia/macrophages in lesion site o Immediate inhibition of CSPG IGF-1 & TNF- -> impaired functional motor recovery & tissue loss o Delayed inhibition improved recovery

 Protein tyrosine phosphatase sigma (PTP ) deficient mice = dont respond

to CSPGs . .. axon regeneration

Semaphorin 3A present in glial scars & contributes to outgrowth inhibitory properties of these scars Ephrin B3 functions through EphA4 receptor and inhibits remyelination NOGO

 Myelin expresses growth inhibitory molecules including: Nogo -A, MAG

&OMgp o IN-1 antibody binds Nogo -A & neutralises inhibitory effects -> CNS axon growth & induced CNS axons to sprout collaterals reported in animal models

Recently shown to promote growth of corticospinal axons in marmoset monkeys

Lee et al. in 2009 claim there is no corticospinal axon regeneration however! Past studies have produced mixed results regarding generative phenotype of Nogo KO mice -> 2 lines didnt show any regeneration & 1 displayed modest regeneration; the 4 th reported extensive regeneration but confounded with an axon la belling artefact

o Peptide inhibitor (NEP1-40) also developed which targets Nogo -A receptor & prevents neurones responding

 Leucine rich repeat domains of the NgR necessary for interaction with
Nogo-A, MAG &OMgp -> GPI anchored protein so recruits co -receptor molecules i.e. p75 (but most adult neurones dont express p75!) o Co-receptor activation (+) Rho & ROCK pathways -> modulates cytoskeleton &neurite growth o NgR-independent cAMP/PKA pathway also affects

Elevation of cAMP by direct injection in DRG overco mes MAG & myelin induced inhibition ->upregulatesArginasel (+) polyamines (+) axonal growth

 Filbrin PirB may be another receptor that responds to myelin inhibitors  NgR1, LINGO-1 & TROY/p75 = shared receptor complex for myelin derived inhibitors (Mandemakers) CELLULAR TRANSPLANTATION

 Bridges cysts& cavities, replaces dead cells, provide new

neurones/myelinating cells, create favourable environment for regeneration

 Work through myelination, neuroprotection, plasticity  Neural progenitors from 8 week old human foetuses transplanted into
immunosuppressed nonhuman primates 9 days after cervical contusion -? spontaneous locomotion within cage & forelimb grip power, no sign of tumour formation cavitation &

 Autologous remain to be tested (but working on how to stimulate patients

endogenous adult progenitors)

 Purified Stem (can differentiate into any cell type

totipotent)/progenitor (can differentiate into many cell types) cells can be collected at 3 diffe rent stages of development: inner cell mass of

blastocyst/ CNS, olfactory system or umbilical cord of fetus/CNS, olfactory system, bone marrow or blood of adult

 Each of these cell populations can be growin in vitro & engineered to

produce a molecule of in terest/restricted to a particular cell fate before transplantation

 Some can be injected directly into the cord (fetus)  Some can be used for autologous transplantation (olfactory
system/umbilical cord blood cells which can be frozen at birth/blood/bone marrow) o Peripheral nerve

Autologous transplants in rats (+) growth of all axonal types but not supraspinal axons/some spinal axons were found to have regenerated 4 months after injury in nonhuman primates

o Schwann cells

  

Bruce et al. human axons remyelinated by Schwann cells Alternatively implant oligodendrocyte precursors Functional recovery and/or CNS axon regeneration when transplanted immediately or up to 2 months after SCI in adult rats

o Cells from embryonic & adult olfactory bulb/mucosa

GROWTH FACTORS

 Following contusion/laceration injury growth factors become

undetectable esp. neurotrophins 3 & 4 or only slightly upregulated& briefly i.e. NGF

 Message levels for neurotrophin receptors i.e. trkA remain absent, trkB&
C remain unaltered, p75 upregu lated o Upon ligand binding, receptors dimerise& p75 signals through Rho but also MAPK pathways etc.

 BDNF may not be available for signalling through trkB however since
there is a sustained upregulation of competitor binding sites

 To promote CNS axon regeneration & functional recovery after SCI,

growth factors have been delivered to soma: o By direct injection o By osmotic mimpump

o By fibrin glue o By gene transfer techniques

 Delivery of these (+) recovery of CNS axons in vivo implantation of NT3 secreting fibroblasts into adult rats caused CST axons to grow up to 8 mm/BDNF-expressing fibroblasts (+) 7% rubrospinal axons regenerated

 Severely injured axons can be rescued by applying BDNF  Gave nonhuman primates NGF firoblasts but need to study functional
recovery in injured animals

 Clinical trials using systemic NTs

- side effects i.e. severe muscular

pain, depression & hallucinations (use viral vectors to target tissues) Epilepsy and anticonvulsants Epilepsy disorder of the CNS characterised by sudden, large increases in electrical activity (seizures) that may be localised/generalised *derives from Greek meaning to seize since ancients believed it was a possession from God Normally spread of electrical activity between neurones is restricted & synchronous discharge is confined to small groups, producing normal EEG rhythms. In seizures, large groups of neurones are activated repetitively (but low frequency), unrestrictedly and hypersynchronously and synaptic inhibition fails producing high oltage spike and wa e acti ity. What causes this? Pre enting burst mode firing: It currents are low-threshold Ca 2+ currents which are activated ~RMP but are simultaneously inactivated to prevent burst mode firing. Following an AP, to prevent summation of excita tion of synaptic inputs I h currents which hyperpolarise the cell are activated. It makes cells more excitable Ih causes greater summation of excitatory inputs Channelopathies: benign familial neonatal convulsions (non -functional potassium channels)/generalised epilepsy with febrile seizures (beta subunits alter inactivation kinetics of alpha subunit in Na + channel -> slower inactivation) Paroxysmal depolarising shift in a single cortical neurone
3 3 3 3

Sustained neuronal depolarisation resulting in a burst of action potentials o Glutamate mediated (AMPA, NMDA)

 

Increased glutamate release and receptors in epileptics Astrocytes (communicate with one another through Ca 2+ signalling) trigger synchronous release of glutamate which acts on multiple neurones

o Ca 2+ influx Plateau-like depolarisation following completion of the action potential burst o Ca 2+ opens voltage-gated Na + channels o Na + influx Rapid repolarisation follow ed by hyperpolarisation o GABA mediated

Decreased GABA release in epileptics

o Glutamate depletion/receptor desensitisation o Cl- influx/K + efflux This PDS is spread to neighbouring pyramidal neurones due a failure of inhibitory feedback though local intern eurons (surround inhibition). ?Gain of inhibition GABA excites: underexpression of KCC2 channels in epilepsy which maintain low intracellular Cl - to ensure efflux upon GABA binding o NKCC1 dominants which pump Cl - in resulting in depolarisation when GABA opens chloride channels GABA agonist drugs provoke seizures in animal models of absence epilepsy GABA makes seizures persist: convulsant (kainite) infused into ipsilateral hippocampus & evoked seizures in both ipsi - & contrao If contra- treated before it became epileptogenic with GABA antagonist bicuculline it didnt stop seizures but if treated after it did EEG (electroencephalography): recording of brains spontaneous electrical activity over 20-40 minutes from multiple electrodes placed on the scalp.

Electrical potentials generated by single neurones too small to be detected so EEG reflects summation of synchronous activity of many neurones with similar spatial orientation o Have to have similar spatial orientations or volume conduction cannot occur

Volume conduction -> local currents generated (ion movement) when synapses activate neighbouring neurones -> attract/repel electrons in metal electrode generating voltage

o Pyramidal neurones produce most of the EEG signal since they are well-aligned and fire together y Limitations: most sensitive to superficial layers of the cortex so cant measure activity of midline/deep structures i.e. hippocampus/meninges and skull smear the EEG signal o Alternatively can use MEG which measures magnetic fields (electrical currents produce magnetic fields) Types of epilepsy y Generalised: affects the whole cortex (impaired consciousness) o Primarily generalised: started at a focal point and s pread out but focal point unknown

 

Absence (petit-mal): sudden brief loss of consciousness; common in children Tonic-clonic (grand-mal): sudden stiffening of muscles following by jerking movements Tonic phase caused by rapid discharge; frequency of discharge slow in clonic o Following seizure (post -ictal phase) discharges still occur but no associated abnormalities Status epilepticus: medical emergency when seizures occur for >30 mins& consciousness not regained (caused by grand -mal)

o Secondarily generalised: started from a specific focal point that can be determined (mostly cortical -> temporal most prevalent esp. hippocampus &entorhinal cortex) y Partial (focal): affects one part of the cortex

o Simple: no loss of consciousness

Jacksonian: focal cortical seizure characterised by jerking movements that begin in limbs and spread to rest of the body

o Complex: impaired consciousness o Aura: stereotyped perception before the seizure (impaired perception after) i.e. smell (visual/gustator y), tingling in one limb or strange inner feelings (dj vu) Treatments for epilepsy 1) Drugs which GABAergic activity a. Benzodiazepines (diazepam) make GABAA receptors more responsive to GABA i. Only used in status epilepticus as it causes amnesia & sedation and myoclonic seizures because they are muscle relaxants b. Barbiturates (phenobarbitone) make Cl - channels open longer c. Vigabatrin x GABA transaminase d. Tiagabine x GABA reuptake 2) Drugs for grand mal a. Carbamapezine, lamogatrin, phenotyin bind inactivated Na + channels & refractory period i. Lamogatrin can be used for petit mal too 3) Drugs for petit mal a. Sodium valproate x Na + channels & GABA transaminase i. Can also be used for grand mal b. Ethosuxamide, gabapentin, pregabilin block T-type Ca 2+ channels i. Ethosuximide doesnt bind Ca 2+ channels at synapses so cant block NT release 4) Atypical anticonvulsants a. Retigabine activates KCNQ2/3 & KCNQ3/5 potassium channels b. Felbamate blocks NMDA receptors c. Topiramate blocks AMPA/kainite receptors d. Levetiracetam blocks fusion of vesicles with membrane e. Losigamone blocks Na + currents on postsynaptic membrane 5) Ketogenic diet (high fat/low carbohydrate -> good for children)

6) Vagal stimulation 7) Surgery to remove the focal point Sodium channel blockers are commonly used in partial seizures