Charles H. Hubscher

Chondroitinase ABC digestion of the perineuronal net promotes functional collateral sprouting in the cuneate nucleus after cervical spinal cord injury.

Upregulation of extracellular chondroitin sulfate proteoglycans (CSPGs) after CNS injuries contributes to the impediment of functional recovery by restricting both axonal regeneration and synaptic plasticity. In the present study, the effect of degrading CSPGs with the application of the bacterial enzyme chondroitinase ABC (chABC) into the cuneate nucleus of rats partially denervated of forepaw dorsal column axons was examined. A dorsal column transection between the C6–C7 dorsal root entry zones was followed immediately by an ipsilateral brainstem injection of either chABC or a bacterial-derived control enzyme [penicillinase (P-ase)] and then subsequently (1 week later) followed with a second brainstem enzyme injection and cholera toxin B subunit (CTB) tracer injection into the ipsilateral forepaw digits and pads. After 1 additional week, the rats underwent electrophysiological receptive field mapping of the cuneate nucleus and/or anatomical evaluation. Examination of the brainstems of rats from each group revealed that CSPGs had been reduced after chABC treatment. Importantly, in the chABC-treated rats (but not in the P-ase controls), a significantly greater area of the cuneate nucleus was occupied by physiologically active CTB traced forepaw afferents that had been spared by the initial cord lesion. These results demonstrate, for the first time, a functional change directly linked to anatomical evidence of sprouting by spinal cord afferents after chABC treatment.

Chronic spinal cord injury induced changes in the responses of thalamic neurons

Sensory disturbances following spinal cord injury (SCI) include chronic pain, which is often localized at spinal levels just rostral to the lesion (referred to as at-level neuropathic pain) and not effectively relieved by traditional treatments. In the present study, a clinically relevant spinal contusion injury was made at the spinal T8 level in 11 deeply anesthetized male rats. Behavioral testing just prior to terminal electrophysiological experiments (done at 30 days post-injury) demonstrated at-level sensitivity to touching the trunk (i.e., allodynia) in 64% of the animals. Electrophysiological data (urethane anesthesia) were obtained for 218 single somatovisceral convergent neurons that were located throughout 12 subregions of the thalamus. In total, 90% (197 of 218) responded to noxious at-level pinch, compared to 52% for pinching the dorsal trunk at the same level in uninjured controls (our previously published data--recorded from 133 total neurons). In addition, 33% of the total neurons tested also responded to gentle touch (dorsal trunk) versus 9% in controls. A comparison of electrophysiological and behavioral data for each individual animal reveals novel tactile neuronal responses within ventral and posterior thalamic subnuclei for those rats showing signs of at-level allodynia. These data suggest that neurons in specific regions of thalamus undergo significant changes in responsiveness following severe chronic SCI. The observed plasticity and ensuing hypersensitivity are likely part of the central reorganization producing the multitude of sensory disturbances that surface following SCI.

Brainstem microstimulation differentially inhibits pudendal motoneuron reflex inputs.

SENSORY information from the genitourinary tract is transmitted to perineal muscle reflex circuits in the spinal cord and to the medullary reticular formation (MRF). This is the first report of a functional descending connection between neurons in the MRF and sensory inputs of the pudendal motoneuron reflex circuitry. In anesthetized rats, microstimulation of the MRF produced a decrease in amplitude and increase in latency of pudendal motoneuron reflex discharges (PMRD) elicited by stimulation of the dorsal nerve of the penis. No effects on pelvic nerve-elicited PMRD were found. The most robust reflex depression was observed when the lateral paragigantocellular reticular nucleus was microstimulated bilaterally. Reproductive abnormalities that occur following severe spinal cord injury probably result from disruption of these descending connections.

Convergence of multiple pelvic organ inputs in the rat rostral medulla

Electrophysiological recordings were used to investigate the degree of pelvic/visceral convergent inputs onto single medullary reticular formation (MRF) neurons. A total of 94 MRF neurons responsive to bilateral electrical stimulation of the pelvic nerve (PN) in 12 urethane‐anaesthetized male rats were tested for responses to mechanical stimulation of the urinary bladder, urethra, colon and penis, and electrical stimulation of the dorsal nerve of the penis (DNP) and abdominal branches of the vagus. Responses to distension of the bladder were found for 51% (n= 48) of the MRF neurons tested. Of these 48, 71% responded to urethral infusion, 81% responded to colon distension, 100% responded to penile stimulation (and DNP), and 85% responded to vagal stimulation, with 62% responding to stimulation of all four of these territories. This high degree of visceral convergence (i.e. 62%) in a subset of PN‐responsive MRF neurons is significantly greater than for the subset of PN‐responsive MRF neurons that did not respond to urinary bladder distension (i.e. out of the 46 remaining neurons, none responded to all four of the other pelvic/visceral stimuli combined). These results suggest that the neurons processing information from the urinary bladder at this level of the neural axis are likely to be important for mediating interactions between different visceral organs for the coordination of multiple pelvic/visceral functions.

Loss and spontaneous recovery of forelimb evoked potentials in both the adult rat cuneate nucleus and somatosensory cortex following contusive cervical spinal cord injury

Varying degrees of neurologic function spontaneously recovers in humans and animals during the days and months after spinal cord injury (SCI). For example, abolished upper limb somatosensory potentials (SSEPs) and cutaneous sensations can recover in persons post-contusive cervical SCI. To maximize recovery and the development/evaluation of repair strategies, a better understanding of the anatomical locations and physiological processes underlying spontaneous recovery after SCI is needed. As an initial step, the present study examined whether recovery of upper limb SSEPs after contusive cervical SCI was due to the integrity of some spared dorsal column primary afferents that terminate within the cuneate nucleus and not one of several alternate routes. C5-6 contusions were performed on male adult rats. Electrophysiological techniques were used in the same rat to determine forelimb evoked neuronal responses in both cortex (SSEPs) and the cuneate nucleus (terminal extracellular recordings). SSEPs were not evoked 2 days post-SCI but were found at 7 days and beyond, with an observed change in latencies between 7 and 14 days (suggestive of spared axon remyelination). Forelimb evoked activity in the cuneate nucleus at 15 but not 3 days post-injury occurred despite dorsal column damage throughout the cervical injury (as seen histologically). Neuroanatomical tracing (using 1% unconjugated cholera toxin B subunit) confirmed that upper limb primary afferent terminals remained within the cuneate nuclei. Taken together, these results indicate that neural transmission between dorsal column primary afferents and cuneate nuclei neurons is likely involved in the recovery of upper limb SSEPs after contusive cervical SCI.

Co-expression of P2X receptor subunits on rat nodose neurons that bind the isolectin GS-I-B4.

Triple fluorescent histochemistry was used to describe the types of overlap in visceral sensory neurons (nodose ganglion) for the labeling of the isolectin B4 from Griffonia simplicifolia type one (GS-I-B4) and their immunoreactivity (IR) for two of the ATP receptor subunits (P2X1/3 or P2X2/3). The vast majority of nodose neurons expressed GS-I-B4-binding and most of these displayed P2X receptor IR. Most of the P2X-IR was co-expressed on these individual nodose neurons (P2X1/P2X3 or P2X2/P2X3). A very small subpopulation of neurons that were GS-I-B4 negative but P2X positive displayed a very high relative intensity of P2X3-IR. The functional role that these expression patterns play in visceral sensory processing is currently unclear.

Brainstem microstimulation activates sympathetic fibers in pudendal nerve motor branch.

A bilateral spino-bulbo-spinal circuit conveys information from/to the male urogenital tract and perineal muscles. This is the first electrophysiological report of another descending pathway, one which conveys output from the medullary reticular formation (MRF) to activate postganglionic sympathetic fibers contained within the motor branch of the pudendal nerve (PudM). In anesthetized rats, long latency (> 150 ms) discharges were elicited in the PudM following ipsilateral or contralateral microstimulation of the MRF. These firing bursts were not observed in rats after sectioning the lower lumbar sympathetic trunk. The most robust activation was observed when neurons in or near the lateral paragigantocellular reticular nucleus were microstimulated bilaterally. Urogenital dysfunction that occurs following severe spinal cord injury probably results from disrupting these and other supraspinal circuits.

Novel Multi-System Functional Gains via Task Specific Training in Spinal Cord Injured Male Rats

Locomotor training (LT) after spinal cord injury (SCI) is a rehabilitative therapy used to enhance locomotor recovery. There is evidence, primarily anecdotal, also associating LT with improvements in bladder function and reduction in some types of SCI-related pain. In the present study, we determined if a step training paradigm could improve outcome measures of locomotion, bladder function, and pain/allodynia. After a T10 contusive SCI trained animals (adult male Wistar rats), trained animals began quadrupedal step training beginning 2 weeks post-SCI for 1 h/day. End of study experiments (3 months of training) revealed significant changes in limb kinematics, gait, and hindlimb flexor-extensor bursting patterns relative to non-trained controls. Importantly, micturition function, evaluated with terminal transvesical cystometry, was significantly improved in the step trained group (increased voiding efficiency, intercontraction interval, and contraction amplitude). Because both SCI and LT affect neurotrophin signaling, and neurotrophins are involved with post-SCI plasticity in micturition pathways, we measured bladder neurotrophin mRNA. Training regulated the expression of nerve growth factor (NGF) but not BDNF or NT3. Bladder NGF mRNA levels were inversely related to bladder function in the trained group. Monitoring of overground locomotion and neuropathic pain throughout the study revealed significant improvements, beginning after 3 weeks of training, which in both cases remained consistent for the study duration. These novel findings, improving non-locomotor in addition to locomotor functions, demonstrate that step training post-SCI could contribute to multiple quality of life gains, targeting patient-centered high priority deficits.

Spinal Cord Injuries Containing Asymmetrical Damage in the Ventrolateral Funiculus Is Associated With a Higher Incidence of At-Level Allodynia

UNLABELLED Approximately 70% of male rats receiving severe T8 spinal contusions develop allodynia in T5-7 dermatomes (at-level) beginning 2 weeks after injury. In contrast, rats having either complete transections or dorsal hemisections do not develop allodynia at-level after chronic spinal cord injury (SCI). In the present study, incomplete laceration and contusion injuries were made to test for neuroanatomical correlates between areas of white matter damage/sparing at the lesion epicenter and the presence/absence of allodynia. After incomplete laceration lesions and 6 weeks of behavioral testing, histological reconstruction and analysis of the lesion epicenters revealed a significant difference (P < .001) in the amount of ventrolateral funiculus (VLF) asymmetry between rats showing pain-like responses evoked by touch (74.5% +/- 8.4% side-to-side difference in VLF damage) versus those not responding to touch (11.3% +/- 4.4% side-to-side difference in VLF damage). A 5-week mean allodynia score for each rat that incorporates a full range of forces that are all innocuous in intact controls revealed that the degree of hypersensitivity at level is related to the extent of VLF asymmetry after SCI. No other damaged spinal white matter or gray matter area was correlated with sensitivity to touch. Similar findings were obtained for rats receiving T8 contusions, a more clinically relevant injury. These data suggest that different extents of damage/sparing between the 2 sides of VLF probably are a requisite for the development of allodynia after SCI. PERSPECTIVE A side-to-side lesion asymmetry after chronic SCI in a rodent model was found to be highly correlated with the presence and degree of allodynia. Greater insight of key factors contributing to the development and maintenance of chronic neuropathic pain is important for improving quality of life.

Sex and hormonal variations in the development of at-level allodynia in a rat chronic spinal cord injury model

The development of central neuropathic pain varies among patients with spinal cord injury (SCI). The factors contributing to the development and perpetuation of segmental pain (at-level allodynia) has been the focus of ongoing experiments in our laboratory. One such factor is hormonal status. We have shown previously, using a male rat model of SCI, that a severe contusion injury is necessary for the development of allodynia in trunk regions at and just above the level of a T8 injury. In this study, we examined at-level sensitivity for SCI ovariectomized (ovx) and cycling female rats as well as for SCI males implanted with either a placebo pellet or one that slowly releases 17beta-estradiol. The proportion of ovx SCI female rats and placebo-treated SCI males displaying pain-like behaviors to touch/pressure of at-level dermatomes up to 6 weeks post-injury (67% and 75%, respectively) was similar to our previous studies on SCI males (69%). In contrast, significantly fewer cycling SCI female rats and 17beta-estradiol treated SCI male rats showed sensitivity to touch at-level (26% and 30%, respectively). These results implicate 17beta-estradiol as a potential target that can readily be modulated to prevent segmental pain following SCI.