Claire E. Hulsebosch

Neocortical neural sprouting, synaptogenesis, and behavioral recovery after neocortical infarction in rats.

BACKGROUND AND PURPOSE Neuroanatomical plasticity is well described in lesions of the hippocampus but remains a subject of some controversy in the neocortex. The purpose of the present study was to measure the neocortical distribution and density of expression of proteins known to be involved in neurite growth or synaptogenesis and to correlate the neocortical expression with behavioral recovery after a focal neocortical infarction. Focal neocortical infarction creates a circumscribed lesion in the neocortex that provides a denervation stimulus for neurite growth and synaptogenesis. METHODS Unilateral neocortical ischemia was induced in male spontaneously hypertensive Wistar rats (n = 4 per time point) by permanent occlusion of the distal middle cerebral artery and ipsilateral common carotid artery. To determine the spatial and temporal distribution of neurite growth and/or synaptogenesis, GAP-43, a growth-associated protein expressed on axonal growth cones, and synaptophysin, a calcium-binding protein found on synaptic vesicles, were examined by immunohistochemical techniques. The reaction product was measured, and the distribution was recorded. Since the resulting infarction included a portion of the forelimb neocortex, behavioral assessments of forelimb function that used the foot-fault test of Hernandez and Schallert were performed on the same rats used for immunohistochemical studies. Recovery times were 3, 7, 14, 30, and 60 days after surgery. RESULTS Both GAP-43 and synaptophysin proteins demonstrated statistically significant increases in the density of immunoreaction product as determined by optical density measurements in the neocortex of infarcted rats compared with sham controls. The GAP-43 was elevated to statistically significant levels in forelimb, hindlimb, and parietal neocortical regions medial and lateral to the infarction only at days 3, 7, and 14. In contrast, synaptophysin demonstrated no statistically significant changes in expression at 3 or 7 days but demonstrated statistically significant increases at 14, 30, and 60 days in the forelimb, hindlimb, and parietal neocortical regions medial and lateral to the infarction as well as in the contralateral parietal neocortex. Behavioral assessment of forelimb function indicated impairment of forelimb placement on the side contralateral to the infarction that trended toward control values at 14 days and was not significantly different from controls by 30 days. CONCLUSIONS These data support the occurrence of neurite growth followed by synaptogenesis in the neocortex, ipsilateral and contralateral to neocortical ischemia, in a pattern that corresponds both spatially and temporally with behavioral recovery. Thus, neuroanatomical remodeling in the neocortex provides a mechanism for recovery of function.

Enhanced Neocortical Neural Sprouting, Synaptogenesis, and Behavioral Recovery With d-Amphetamine Therapy After Neocortical Infarction in Rats

BACKGROUND AND PURPOSE D-Amphetamine administration increases behavioral recovery after various cortical lesions including cortical ablations, contusions, and focal ischemia in animals and after stroke in humans. The purpose of the present study was to test the enhanced behavioral recovery and increased expression of proteins involved in neurite growth and synaptogenesis in D-amphetamine-treated rats compared with vehicle-treated controls after a focal neocortical infarct. METHODS Unilateral neocortical ischemia was induced in male spontaneously hypertensive Wistar rats (n=8 per time point per group) by permanently occluding the distal middle cerebral artery and ipsilateral common carotid artery in 2 groups of rats: D-amphetamine treated (2 mg/kg IP injections) and vehicle treated (saline IP injections). To determine the spatial and temporal distribution of neurite growth and/or synaptogenesis, growth-associated protein (GAP-43), a protein expressed on axonal growth cones, and synaptophysin, a calcium-binding protein found on synaptic vesicles, were examined by immunohistochemical techniques, and both density and distribution of reaction product were measured. Since the resulting infarction included a portion of the forelimb neocortex, behavioral assessments of forelimb function using the foot-fault test of Hernandez and Schallert were performed on the same rats used for immunohistochemical studies during the period of drug action and 24 hours later. A Morris water maze and other indices of behavioral assays were also measured similarly. Recovery times were 3, 7, 14, 30, and 60 days postoperatively. RESULTS Both GAP-43 and synaptophysin proteins demonstrated statistically significant increases in density and distribution of immunoreaction product as determined by optical density measurements in the neocortex of the infarcted group treated with D-amphetamines compared with vehicle-treated infarcted controls. The GAP-43 was elevated to statistically significant levels in forelimb, hindlimb, and parietal neocortical regions ipsilateral to the infarction only at days 3, 7, and 14. By contrast, the synaptophysin demonstrated no statistically significant changes in expression at 3 or 7 days but demonstrated statistically significant increases at 14, 30, and 60 days in the forelimb, hindlimb, and parietal neocortical regions ipsilateral to the infarction as well as increased distribution in the contralateral parietal neocortex. Behavioral assessment of forelimb function indicated that improved recovery of forelimb placement on the side contralateral to the infarction was statistically significant in the D-amphetamine-treated group compared with the vehicle-treated group (P<0.025). Spatial memory, as measured with the Morris water maze, worsened in the vehicle-treated group compared with the D-amphetamine-treated group at 60 days (P<0.025). CONCLUSIONS These data support the occurrence of neurite growth followed by synaptogenesis in the neocortex in a pattern that corresponds both spatially and temporally with behavioral recovery that is accelerated by D-amphetamine treatment. While the specific mechanisms responsible for D-amphetamine-promoted expression of proteins involved in neurite growth and synaptogenesis and of enhanced behavioral recovery are not known, it is suggested that protein upregulation occurs as a result of functional activation of pathways able to remodel in response to active behavioral performance.

Mechanical and thermal allodynia in chronic central pain following spinal cord injury

&NA; Spinal cord injury (SCI) results in variable motor recoveries and chronic central pain syndromes develop in the majority of SCI patients. To provide a basis for further studies, we report a new rodent model of chronic central pain following spinal cord trauma. Male Sprague‐Dawley rats (N = 10) were hemisectioned at T13 and were tested both preoperatively and postoperatively and compared to sham‐operated controls (N = 10) for locomotor function, and mechanical and thermal thresholds of both paw withdrawal and supraspinal responses. Results support the development and persistence of allodynia which persists for 160 days. Locomotor function was tested using the Basso, Beattie and Bresnahan (BBB) open field test and only the limb ipsilateral to the hemisection was affected, demonstrating acute flaccid paralysis with motor recovery which approached normal values by postoperative day (POD) 15. Prior to the hemisection, the rats showed little to no paw withdrawal response to von Frey stimulation of 4.41 mN or 9.41 mN in both forelimbs and hindlimbs. Postoperatively, responses in both ipsilateral and contralateral forelimbs and hindlimbs increased over time and the increase was statistically significant compared to intra‐animal presurgical and sham control values (P < 0.05). There were no significant side‐to‐side differences in limb responses preoperatively or beyond POD 15. The forelimbs and hindlimbs responded to von Frey hair strengths of 122 mN preoperatively and postoperatively with similar withdrawal frequencies that were not statistically significant. Preoperatively, the paw withdrawal latency to heat stimuli was 22.9 ± 3.0 (mean ± SE) and 20.1 ± 3.1 sec for the hindlimbs and forelimbs, respectively. Postoperatively, the mean hindlimb and forelimb latency of paw withdrawals decreased to 11.9 ± 1.8 and 9.2 ± 2.5 sec, respectively. This decrease in thermal thresholds is statistically significant when compared to intra‐animal preoperative and sham control values (P < 0.05). These data indicate that somatosensory thresholds for non‐noxious mechanical and radiant heat which elicit paw withdrawal (flexor reflex) are significantly lowered following SCI. To further support the development and persistence of chronic pain following hemisection, supraspinal responses such as paw lick, head turns, attacking the stimulus, and vocalizations were elicited in response to mechanical and thermal stimuli and were statistically significant compared to presurgical intra‐animal or sham control values (P < 0.05). Hemisected animals vocalized to von Frey hair bending forces of 49.8 with a mean of 6.0 ± 1.2 times out of 10 stimuli compared to intra‐animal presurgical and sham control values of zero. Supraspinal responses of hemisected animals to thermal stimuli occurred at lower temperatures that were statistically significant compared to sham control or preoperative values (P < 0.05). These chronic changes in thresholds to both mechanical and thermal stimuli represent the development and persistence of mechanical and thermal allodynia after SCI.

Spinal Cord Injury and Anti-NGF Treatment Results in Changes in CGRP Density and Distribution in the Dorsal Horn in the Rat

Spinal cord injury (SCI) results in chronic pain states in which the underlying mechanism is poorly understood. To begin to explore possible mechanisms, calcitonin gene-related peptide (CGRP), a neuropeptide confined to fine primary afferent terminals in laminae I and II in the dorsal horn of the spinal cord and implicated in pain transmission, was selected. Immunocytochemical techniques were used to examine the temporal and spatial distribution of CGRP in the spinal cord following T-13 spinal cord hemisection in adult male Sprague-Dawley rats compared to that seen in sham controls. Spinal cords from both hemisected and sham control groups (N = 5, per time point) were examined on postoperative day (POD) 3, 5, 7, 14, and 108 following surgery. Sham operated rats displayed CGRP immunoreaction product in laminae I and II outer, Lissauers tract, dorsal roots, and motor neurons of the ventral horn. In the hemisected group, densiometric data demonstrated an increased deposition of reaction product that was statistically significant, in laminae III and IV, both ipsilateral and contralateral to the lesion that extended at least two segments rostral and caudal to the hemisection site by POD 14, and remained significantly elevated as long as POD 108. Since upregulation alone of CGRP would occur in an acute temporal window (by 2 to 3 days following spinal injury), these results are interpreted to be invasion of laminae III and IV by sprouting of CGRP containing fine primary afferents. Intrathecal delivery of antibodies against purified 2.5S nerve growth factor for 14 days to the hemisected group resulted in CGRP density in laminae I through IV that was significantly less than that seen in untreated or vehicle treated hemisected groups and to sham controls. These data indicate changes in density and distribution of CGRP following spinal hemisection that can be manipulated by changes in endogenous levels of NGF. These observations suggest possible strategies for intervention in the development of various pain states in human SCI.

Alleviation of mechanical and thermal allodynia by CGRP8-37 in a rodent model of chronic central pain

&NA; CGRP8‐37 is a truncated version of calcitonin gene‐related peptide (CGRP) that binds to the CGRP receptor with similar affinity but does not activate the receptor and is a highly selective CGRP receptor antagonist. CGRP and activation of its receptor appear to play a role in peripheral inflammatory and neuropathic models of pain although there is considerable controversy. The aim of this study was to examine possible anti‐nociceptive effects of CGRP8‐37 on a model of chronic central neuropathic pain known to develop weeks after spinal hemisection. Adult male Sprague–Dawley rats were given a spinal hemisection (N=34) or a sham surgery (N=10) at the T13 spinal segment. An externally accessible PE‐10 intrathecal catheter that terminated at T13 was used for drug delivery. Animals were allowed to recover for 4 weeks at which time the hemisected animals displayed mechanical and thermal allodynia bilaterally, in both forelimbs and hindlimbs. CGRP8‐37 was delivered just prior to a testing session in 1, 5, 10, or 50 nM doses in artificial cerebral spinal fluid in 10 &mgr;l volumes. CGRP8‐37 was effective in alleviating mechanical and thermal allodynia in a dose‐dependent manner (P<0.05). The 50 nM dose was most efficacious for both forelimb and hindlimb responses (P<0.05). The period of efficacy was 10 min to onset for a duration of 20 min. Post‐drug washout responses were not statistically significant compared to pre‐drug responses. The sham control groups demonstrated no statistically significant difference at any dose of CGRP8‐37 when compared to pre‐surgical baseline values. In conclusion, CGRP8‐37 is effective in abolishing mechanical and thermal allodynia produced by spinal hemisection. Consequently, the CGRP receptor may play a role in chronic central neuropathic pain and offers a novel therapeutic approach to managing chronic central pain.

IL-1 Receptor Antagonist Prevents Apoptosis and Caspase-3 Activation after Spinal Cord Injury

One of the consequences of cytokine-orchestrated inflammation after CNS trauma is apoptosis. Our hypothesis is that cell death in the spinal cord after injury results in part from increased synthesis and release of IL-1beta. Using a ribonuclease protection assay, we demonstrated that there is increased transient expression of IL-1beta mRNA and, by using IL-1beta protein ELISA assay, that there are increased IL-1beta protein levels in the contused rat spinal cord, initially localized to the impact region of the spinal cord (segment T8). Using an ELISA cell death assay, we showed that there is apoptosis in the spinal cord 72 h after injury, a finding that was confirmed by measuring caspase-3 activity, which also significantly increased at the site of injury 72 h after trauma. Treatment of the contused spinal cord at the site of injury with the IL-1 receptor antagonist (rmIL-lra, 750 ng/mL) for 72 h using an osmotic minipump completely abolished the increases in contusion-induced apoptosis and caspase-3 activity.

Changes in serotonin, serotonin transporter expression and serotonin denervation supersensitivity: involvement in chronic central pain after spinal hemisection in the rat.

Spinal cord injury (SCI) results in abnormal locomotor and pain syndromes in humans. In a rodent SCI model, T13 unilateral spinal hemisection results in bilateral mechanical allodynia and thermal hyperalgesia, partly by interruption of tonic descending serotonin (5-HT) inhibition. In the current study, we examined changes in density and distribution of 5-HT and 5-HT(T) in cervical (C8) and lumbar (L5) enlargements after T13 spinal hemisection and studied the effects of intrathecally delivered 5-HT (10, 21, and 63 microg), 5-HT antagonist methysergide (125 microg/kg), and 5-HT reuptake inhibitor fluvoxamine (75 microg/kg) on pain-related behaviors. Thirty-day-old male Sprague-Dawley rats were spinally hemisected and sacrificed at 3 (n = 20) and 28 (n = 20) days postsurgery for immunohistochemistry, Western blot, and ELISA analysis and compared against sham-operated animals (n = 10). At day 3, C8 5-HT levels were not significantly changed but at L5 there was a significant decrease in ipsilateral 5-HT in laminae I-II followed by incomplete recovery at 28 days postinjury. At both 3 and 28 days postinjury, C8 5-HT(T) levels were not significantly changed, but at L5 there was significant ipsilateral up-regulation of 5-HT(T) in laminae I-II. A second group of animals (n = 30) was hemisected and, starting at 28 days postinjury, behaviorally tested with intrathecal compounds. Increasing doses of 5-HT attenuated both fore- and hindlimb mechanical allodynia and thermal hyperalgesia, and effects of endogenous 5-HT were attenuated by methysergide and enhanced with fluvoxamine, all without locomotor alterations. Sham controls (n = 10) were unaffected. Thus, permanent changes occur in 5-HT and 5-HT(T) after SCI, denervation 5-HT supersensitivity develops, and modulation of 5-HT attenuates pain-related behaviors. Insight gained by these studies may aid in the understanding of dynamic 5-HT systems which will be useful in treating chronic central pain after SCI.

Increases in the activated forms of ERK 1/2, p38 MAPK, and CREB are correlated with the expression of at-level mechanical allodynia following spinal cord injury.

Rats given moderate spinal cord injury (SCI) display increases in the expression of the activated form of the transcription factor cyclic AMP responsive element binding protein (CREB) in spinal segments of dermatomes corresponding to permanent mechanical allodynia, a model of chronic central neuropathic pain (CNP; (Crown, E.D., Ye, Z., Johnson, K.M., Xu, G.Y., McAdoo, D.J., Westlund, K.N., Hulsebosch, C.E., 2005. Upregulation of the phosphorylated form of CREB in spinothalamic tract cells following spinal cord injury: relation to central neuropathic pain. Neurosci. Lett. 384, 139-144)). Given that not all rats that receive moderate SCI develop CNP, the current study was designed to further analyze changes in persistent CREB activation and in the activation state of upstream intracellular signaling cascades (e.g., mitogen-activated protein kinases [MAPKs]) in populations of rats that receive SCI and weeks later develop CNP and rats that receive SCI but do not develop CNP. The results indicate that activated kinases such as pERK 1/2, p-p38 MAPK, but not pJNK, are upregulated in injured rats that develop CNP as compared to injured rats that fail to develop CNP. In addition, the current results replicated our previous finding that activated CREB is upregulated following SCI, however, only in SCI rats that developed CNP. Taken together, these results indicate that activation of intracellular signaling cascades traditionally associated with long-term potentiation and memory is associated with the expression of chronic CNP following SCI.

Peripheral and central sensitization in remote spinal cord regions contribute to central neuropathic pain after spinal cord injury

ABSTRACT Central neuropathic pain (CNP) developing after spinal cord injury (SCI) is described by the region affected: above‐level, at‐level and below‐level pain occurs in dermatomes rostral, at/near, or below the SCI level, respectively. People with SCI and rodent models of SCI develop above‐level pain characterized by mechanical allodynia and thermal hyperalgesia. Mechanisms underlying this pain are unknown and the goals of this study were to elucidate components contributing to the generation of above‐level CNP. Following a thoracic (T10) contusion, forelimb nociceptors had enhanced spontaneous activity and were sensitized to mechanical and thermal stimulation of the forepaws 35 days post‐injury. Cervical dorsal horn neurons showed enhanced responses to non‐noxious and noxious mechanical stimulation as well as thermal stimulation of receptive fields. Immunostaining dorsal root ganglion (DRG) cells and cord segments with activating transcription factor 3 (ATF3, a marker for neuronal injury) ruled out neuronal damage as a cause for above‐level sensitization since few C8 DRG cells expressed AFT3 and cervical cord segments had few to no ATF3‐labeled cells. Finally, activated microglia and astrocytes were present in thoracic and cervical cord at 35 days post‐SCI, indicating a rostral spread of glial activation from the injury site. Based on these data, we conclude that peripheral and central sensitization as well as reactive glia in the uninjured cervical cord contribute to CNP. We hypothesize that reactive glia in the cervical cord release pro‐inflammatory substances which drive chronic CNP. Thus a complex cascade of events spanning many cord segments underlies above‐level CNP.

Direct evidence of primary afferent sprouting in distant segments following spinal cord injury in the rat: colocalization of GAP-43 and CGRP

Mechanical and thermal allodynia develops after spinal cord injury in three areas relative to the lesion: below level, at level, and above level. The present study tests colocalization of CGRP, associated with nociceptive neurons, with growth-associated protein (GAP-43), expressed in growing neurites, to test for neurite sprouting as a mechanism for reorganization of pain pathways at the level of the lesion and distant segments. Male Sprague-Dawley rats were divided into three groups: sham control (N = 10), hemisected at T13 and sacrificed at 3 days (N = 5) and at 30 days (N = 5) following surgery, the spinal cord tissue was prepared for standard fluorescent immunocytochemistry using mouse monoclonal anti-GAP-43 (1:200) and/or rabbit polyclonal anti-CGRP (1:200), density of immunoreaction product (IR) was quantified using the Bioquant software and values from the hemisected group were compared to similar regions from the sham control. We report significant increases at C8 and L5, in CGRP-IR in lamina III compared to control tissue (P < 0.05). We report significant bilateral increases in GAP-43-IR at C8, T13, and L5 segments in lamina I through IV, at 3 days post hemisection, compared to control tissue (P < 0.05), some of which is colocalized with alpha-CGRP. The increased area and density of GAP-43-IR is consistent with neurite sprouting, and the colocalization with alpha-CGRP indicates that some of the sprouting neurites are nociceptive primary afferents. These data are consistent with endogenous regenerative neurite growth mechanisms that occur near and several segments from a spinal lesion, that provide one of many substrates for the development and maintenance of the dysfunctional state of allodynia after spinal cord injury.