Gary V. Allen

Conditioning effects of repetitive mild neurotrauma on motor function in an animal model of focal brain injury.

A weight drop model of brain injury was used to determine the effects of repetitive mild brain injury on motor function, heat shock protein and glial fibrillary acidic protein expression in the anesthetized, adult male, Sprague-Dawley rat. Repetitive mild brain injury was produced when animals received a series of three mild injuries spaced three days apart. A separate group of repetitive mild injured animals also received a subsequent severe brain injury between three and five days after the last mild injury. All animals were trained on a beam-walking test prior to surgery. The mild, repetitive mild and repetitive mild plus severe brain injury groups showed no motor deficits in the beam-walking test, whereas the animals with only severe brain injury showed significant motor deficits (increase in number of footslips) in the beam-walking test that recovered within eight days after injury. Both repetitive mild plus severe injury and severe injury only animals had cortical necrotic cavities of similar size in the region of the hindlimb motor cortex. Both the repetitive mild and severe brain-injured animals had marked heat shock protein 27kDa and glial fibrillary acidic protein staining in the cerebral cortex. Fluoro-Jade, heat shock protein 27kDa and 72kDa labeling indicated that there were widespread effects on cortical, subcortical and spinal neurons and glial cells after repetitive mild brain injury. These results suggest that repetitive mild brain injury conditions the brain so that subsequent brain injury at the same site has no effect on motor function. Furthermore, repetitive mild injury-induced activation of processes distant to the primary injury site may have a role in activation of secondary sites involved in recovery of motor function.

Trigeminal-parabrachial connections : possible pathway for nociception-induced cardiovascular reflex responses

Noxious stimulation of dental nerves elicits marked changes in cardiovascular function. In order to investigate central pathways mediating reflex changes in cardiovascular activity, immunohistochemical localization of cells expressing the immediate-early gene, c-fos, was used to identify central nervous responding to noxious electrical stimulation of mandibular, incisor tooth dentin or chemical (capsaicin) stimulation of tooth pulp in the anesthetized rat. Injections of Fluoro-Gold were made in the lateral parabrachial region to identify efferent projections from the spinal trigeminal nucleus. Electrical and chemical stimulation produced similar patterns of Fos-positive neurons in the spinal trigeminal nucleus: subnuclei caudalis, interpolaris and oralis. Fos-positive neurons were most dense in laminae I and II of the dorsomedial subnucleus caudalis with fewer Fos-positive neurons located in the interpolaris and oralis subnuclei. Sham stimulation of tooth dentin and control vehicle injections into the tooth pulp resulted in either a few weakly stained or no Fos-positive neurons in the spinal trigeminal nucleus. Cell bodies double labeled with Fluro-Gold following injections into the parabrachial region and Fos-protein subsequent to electrical stimulation of incisor tooth were present in all three subnuclei of the spinal trigeminal nucleus. The largest number of Fos-positive neurons with efferent projections to the lateral parabrachial region were located in subnucleus caudalis (32.2 +/- 5.3 S.E.M.) and fewer were located in the interpolaris (0.4 +/- 0.4 S.E.M.) and oralis (19.8 +/- 3.5 S.E.M.) subnuclei. The results demonstrate that nociceptive dental input received by the three subnuclei of the spinal trigeminal nucleus, particularly the subnucleus caudalis, is relayed to the lateral parabrachial nucleus.

Chronic administration of amitriptyline and caffeine in a rat model of neuropathic pain: multiple interactions

This study was designed to determine (1) whether chronic amitriptyline administration was effective in alleviating symptoms of neuropathic pain in a rat model of spinal nerve injury, and (2) whether the effect of amitriptyline involved manipulation of endogenous adenosine, by determining the effect of caffeine, a non-selective adenosine A(1) and A(2) receptor antagonist, on its actions. Nerve injury was produced by unilateral spinal nerve ligation of the fifth and sixth lumbar nerves distal to the dorsal root ganglion, and this resulted in stimulus-evoked thermal hyperalgesia and static tactile mechanical allodynia. Animals received pre- and post-surgical intraperitoneal doses of amitriptyline (10 mg/kg) and caffeine (7.5 mg/kg), alone or in combination, and following surgery, were administered amitriptyline (15-18 mg/kg/day) and caffeine (6-8 mg/kg/day), alone or in combination, in the drinking water. Rats were tested for thermal reaction latencies and static tactile thresholds at 7, 14 and 21 days following surgery. In the paw ipsilateral to the nerve ligation, chronic amitriptyline administration consistently decreased the thermal hyperalgesia produced by spinal nerve ligation over a 3-week period, and this effect was blocked by concomitant caffeine administration at all time intervals. In the contralateral paw, thermal withdrawal latencies were more variable, with the most reproducible finding being a reduction in thermal thresholds in the amitriptyline-caffeine combination group. There was no effect by either drug or the drug combination on the static tactile allodynia produced by spinal nerve ligation in the ipsilateral paw. However, chronic amitriptyline administration induced a tactile hyperaesthesia in the contralateral paw at all time intervals, and this effect was exacerbated by concomitant chronic caffeine administration. The results of this study indicate that chronic administration of amitriptyline is effective in alleviating thermal hyperalgesia, but not static tactile allodynia, in the hindpaw ipsilateral to nerve injury, and the block of this effect by caffeine suggests that this effect is partially achieved through manipulation of endogenous adenosine systems. Additionally, chronic amitriptyline administration induces contralateral hyperaesthetic responses that are augmented by caffeine. Both the symptom-specific effect, and adenosine involvement in amitriptyline action may be important considerations governing its use in neuropathic pain.

Trigeminal autonomic pathways involved in nociception-induced reflex cardiovascular responses

Reflex cardiovascular responses elicited by noxious oro-facial stimulation are well known but the neural pathways that underlie trigeminal cardiovascular reflex reactions remain to be elucidated. In previous studies, we have shown that noxious electrical stimulation of the mandibular incisor in the anesthetized rat elicits increases in mean arterial blood pressure and heart rate (Allen, G.V., Barbrick, B. and Esser, M.J., Trigeminal parabrachial connections: possible pathway for nociception-induced cardiovascular reflex responses, Brain Res., 715 (1996) 125-135). In this study, microinjections of the presynaptic blocker, cobalt chloride, or the anesthetic agent, lidocaine, were made into selected brainstem sites to identify neural pathways that are involved in mediation of the reflex pressor responses. Ipsilateral and bilateral injections of chemical blocker into the dorsomedial spinal trigeminal nucleus, pars caudalis, lateral parabrachial nucleus and the rostral ventral lateral medulla/caudal A5 region attenuated the reflex cardiovascular response. Bilateral injections of cobalt chloride into the dorsomedial subnucleus caudalis resulted in 70-100% attenuation of the reflex pressor response. Bilateral injections of cobalt chloride and/or lidocaine into the lateral parabrachial nucleus or the rostral ventral lateral medulla/A5 region resulted in 43-57% and 44-100% attenuation of the reflex pressor response, respectively. There were no significant differences in the degree or duration of attenuation of the reflex pressor responses produced by cobalt chloride compared to that produced by lidocaine injections. The reflex pressor responses usually returned to baseline levels approximately 60 min following injection of the chemical blocker substance. The results indicate that noxious electrical stimulation of the mandibular incisor elicits a reflex increase in mean arterial blood pressure which is initially mediated in the dorsomedial spinal trigeminal nucleus, pars caudalis and is subsequently mediated in the lateral parabrachial nucleus and the rostral ventral lateral medulla/caudal A5 region.

Micturition evoked by glutamate microinjection in the ventrolateral periaqueductal gray is mediated through Barrington’s nucleus in the rat

Neural tracing experiments have demonstrated a direct spinal projection to Barringtons nucleus and a possible indirect pathway to Barringtons nucleus via the periaqueductal gray. We sought to identify the role of the periaqueductal gray matter in micturition in urethane-anesthetized rats. Blockade of micturition by focal injection of cobalt chloride was used to identify sites critical to micturition. These sites were located near the ventral margin of the caudal ventrolateral periaqueductal gray and in Barringtons nucleus. L-Glutamate injections into caudal regions of the periaqueductal gray evoked bladder contraction with coordinated sphincter activation. Additional L-glutamate sites with a similar pattern of response and sites where sphincter activation was produced without bladder contraction were found more rostrally and dorsally in the periaqueductal gray. Activation of bladder contractions by L-glutamate injection in the ventrolateral periaqueductal gray was blocked by prior injection of cobalt chloride into Barringtons nucleus. From these data we propose that ventrolateral periaqueductal gray is functionally important to micturition in the urethane-anesthetized rat. Further, we have shown that a periaqueductal gray to Barringtons nucleus pathway is functionally relevant to central mediation of bladder contraction.

Volume-evoked micturition reflex is mediated by the ventrolateral periaqueductal gray in anesthetized rats

The central pathway of the micturition reflex in the rat was investigated functionally by acute blockade of synaptic neurotransmission using microinjection of cobalt chloride into the periaqueductal gray or pontine tegmental region. In 27 urethan-anesthetized (1.2 g/kg ip) rats, the bladder pressure response to continuous infusion of the bladder with saline (0.1-0.25 ml/min) was assessed. Electromyographic activity of external urethral sphincter and arterial blood pressure were also monitored. Bladder contractions and external urethral sphincter activity were reversibly attenuated after unilateral or bilateral stereotaxic injections of 10 mM cobalt chloride into the caudal (bregma -7.80 to -8.80) ventrolateral periaqueductal gray as well as into Barringtons nucleus. Blood pressure was not affected by injection into either area. The results demonstrate that the caudal ventrolateral periaqueductal gray, in addition to Barringtons nucleus, is a critical part of the long-routed micturition reflex circuitry in the anesthetized rat.

Transnuclear transport and axon collateral projections of the mamillary nuclei in the rat

Transnuclear transport of horseradish peroxidase (HRP) and wheat germ agglutinin-HRP conjugate (WGA-HRP) and the retrograde transport of fluorescent tracers were used to study axon collaterals of neurons in the mamillary nuclei. Tracers were injected into the thalamus or brain stem and after 18 hour-5 day survival periods, the brains were processed for fluorescence microscopy or for light and electron microscopic HRP histochemistry. Neurons in all divisions of the ipsilateral mamillary nuclei projected to both the thalamus and tegmentum. After HRP and WGA-HRP injections, anterogradely labeled axon terminals were observed in the known projection fields of the mamillary nuclei. Mamillary neurons were characterized by deeply invaginated, eccentrically located nuclei. Most labeled terminals of axon collaterals in the contralateral anterodorsal thalamic nucleus and dorsal and ventral tegmental nuclei contained round vesicles and formed asymmetrical synapses with somata and dendrites. The present results demonstrate that transnuclear transport of HRP and WGA-HRP can be used to study the connectivity and ultrastructure of axon collaterals and their cells of origin in the central nervous system in a manner comparable to that of transganglionic transport in the peripheral nervous system.

Trigeminal-reticular connections: possible pathways for nociception-induced cardiovascular reflex responses in the rat.

Cardiovascular regulatory neurons of the ventral medulla and pons are thought to have an important role in the mediation of trigeminal nociception‐induced reflex cardiovascular responses. However, the neural pathways that link the spinal trigeminal nucleus with ventral medullary and pontine autonomic cell groups are poorly understood. The present study utilized injections of the highly sensitive anterograde tracer substance biotinylated dextran combined with immunocytochemistry for tyrosine hydroxylase, the synthesizing enzyme for catecholamines, to investigate the distribution and morphology of projections from the spinal trigeminal subnucleus caudalis to ventral medullary and pontine catecholaminergic cell groups. Injection of biotylinated dextran into the dorsal subnucleus caudalis produced dense anterograde labeling in dorsal regions of the medullary and pontine reticular formation including the dorsal medullary reticular field, the parvicellular reticular field, and the parvicellular reticular field pars anterior. In the ventral medullary and pontine reticular formation, light anterograde labeling tended to be distributed in close proximity to the distal dendrites of catecholaminergic neurons located in the C1, A1, and A5 regions.

A review of heat shock protein induction following cerebellar injury

Exposure of cells to stressful environments such as heat shock, ischemia, trauma and disease, induces the cellular expression of heat shock proteins (Hsps). Since the discovery of heat shock proteins in the early 1960s, efforts to understand their function in both stressed and non-stressed cells have remained the focus of a vast collection of researchers. Post-injury heat shock protein induction is believed to identify regions of reversible cell injury as well as contribute to repair and protective mechanisms following stress. With the role of cerebellum expanding to include a number of cognitive processes in addition to contributing to motor coordination, research contributions that further our understanding of cerebellar repair strategies following injury are significant. Following cellular stress, heat shock protein expression was observed in both neuronal and glial cell populations in the injured cerebellum. Specifically, Hsp27 expression was localized primarily in Purkinje cells and glial cells within the injured cerebellum, whereas Hsp72 induction was more prominent in the granule cell layer of the cerebellum. Thus, there appears to be a preferential expression of different families of heat shock proteins in different cell populations in the injured cerebellum. There are also distinct post-injury time frames of induction for each family of heat shock protein, emphasizing differences in cellular functional requirements for each family of heat shock protein. Hsp27 was expressed immediately following injury and continued up to 20 days post-injury whereas Hsp72 was expressed immediately following injury and disappeared by 4 days post-injury, suggesting the latter contributes to processes involved in the initial repair of injured cells. This review discusses heat shock protein induction patterns in both in vivo and in vitro cerebellar injury models and provides suggestions as to the functional role of heat shock proteins in the injured cerebellum.

Peripheral Amitriptyline Suppresses Formalin-Induced Fos Expression in the Rat Spinal Cord

We examined the effects of systemically, spinally, and peripherally administered amitriptyline on formalin-induced Fos immunoreactivity in the lumbar spinal cord. Formalin (2.5%), injected subcutaneously into the rat hindpaw, increased Fos immunoreactivity in laminae I–II, III–IV, and V–VI of the dorsal L5 spinal cord. Amitriptyline, administered both systemically and spinally before formalin, increased flinching and concurrently decreased biting/licking behaviors, but neither route of administration produced any statistically significant change in Fos immunoreactivity. Amitriptyline coadministered with the formalin reduced both flinching and biting/licking behaviors, and significantly reduced Fos immunoreactivity, particularly in laminae I–II. These immunohistochemical changes reflect the net behavioral effects observed after the different routes of drug administration. The profile of amitriptyline action after peripheral administration may be of clinical importance because of the potential use of antidepressants as topical analgesics.