Hemisection Group Research Articles

A Cadaveric Biomechanical Comparison of a Triple vs Quintuple Hemi-Section Achilles Tendon Lengthening

Category: Ankle; Hindfoot Introduction/Purpose: Achilles tendon lengthening is commonly performed percutaneously, and the triple hemi-section step cut technique proposed by Hoke (1) is the method most commonly used. Despite its popularity, Hoke’s technique has been found to have limited lengthening capacity compared to the traditionally used open lengthening technique and therefore is only used in treating mild Achilles contracture. The senior author (MSM) developed a 5-step (quintuple) percutaneous hemi-section technique for lengthening, demonstrated to be a reliable procedure. The purpose of this cadaveric study was to evaluate the efficacy and safety of the quintuple compared to the triple cut technique. Methods: Four matched pairs of above-knee specimens were randomly assigned to the triple and the other to the quintuple hemi-section group. The Achilles insertion and musculotendinous junction were fully exposed to ensure that tenotomies were performed in a standardized manner. Lengthening was measured with optical markers at the most distal cut and 15 cm proximally. Measurements were performed on a universal testing machine (Instron) with the forefoot and femur fixed, and the knee in extension (Figure 1). The ankle was dorsiflexed under a constant load to 45N. Lengthening was performed 1 cm above the Achilles insertion following a medial-lateral-medial order at 3 cm intervals. The load was gradually increased until maximum dorsiflexion. The dorsiflexion angle, the tendon lengthening, the load needed to achieve the maximum ankle dorsiflexion and any rupture of the Achilles was recorded. Two-sample t-test was used for comparing continuous variables. Statistical analysis was performed with SAS 9.4. Results: There was no statistical difference between the dorsiflexion angle of the two groups under 45N load with an intact Achilles. The quintuple procedure achieved more lengthening than the triple group, reflected in both the maximum dorsiflexion angle under 45N and the lengthening of the tendon between the optical markers. The quintuple technique produced an average of 34.41mm of lengthening, and the triple cut 11.36mm under 45N force. The quintuple technique produced an average of 16.58 degrees of dorsiflexion compared with the triple cut of 10.53 degrees under 45N force. The load required to reach the maximum ankle dorsiflexion in the quintuple group was less (222.9N) than that in the triple group (334.6N). There were no ruptures of the Achilles in the quintuple group and 2 tendon ruptures in the triple technique. Conclusion: The quintuple lengthening is safe and has an increased capacity for tendon lengthening, an advantage over the triple hemi-section technique. Excessive force on the foot attempting to gain more dorsiflexion using the triple cut technique can be avoided with the quintuple procedure in all cases, particularly when adequate dorsiflexion cannot be obtained. References Hoke M. An operation for the correction of extremely relaxed flat feet. J Bone Joint Surg Am. 1931;13:773-783. Hoefnagels EM, Waites MD, Belkoff SM, Swierstra BA. Percutaneous Achilles tendon lengthening: a cadaver-based study of failure of the triple hemisection technique. Acta Orthop. 2007 Dec;(6):808-12

Mechanism of forelimb motor function restoration in rats with cervical spinal cord hemisection-neuroanatomical validation-

PurposeThe purpose of this study is neuroanatomical validation of forelimb motor function restoration in rats with cervical spinal cord injury. Materials and methodsWe used eight cervical hemisected rats and eight normal rats. We cut in half the C3/4 cervical spinal cord of 18-weeks-old normal rats. We used 24-weeks-old rats that had reached a nearly steady state of forelimb motor function after the hemisection (Hemisection group). Normal 24-week-old rats were used as Control group. To evaluate the corticospinal tracts, neuro-tracing by biotynirated dextran-amine (BDA) was used. BDA was injected into the damaged side of the cerebral primary motor cortex. In order to quantitatively analyze the specimen, we recorded a site where nerve fibers appear in each specimen in the image analysis (1) and defined the increase rate of immunostaining area using ImageJ in the image analysis (2). Based on the evaluation in the image analysis (1) and the image analysis (2), the Hemisection group and the Control group were compared. ResultsIn the image analysis (1), a region with robust appearance of aberrant nerve fibers was observed in the cephalad side of the Hemisection site in Hemisection group than Control group. In the spinal cord caudal to the hemisection, such region was generally more in Hemisection group, however, disappeared or reduced appearance was observed in some regions. In the image analysis (2), no statistical significant difference was noted in each level. ConclusionThere is a high probability that these aberrant nerve fibers beyond the midline could be involved in forelimb motor function restoration in rats with cervical cord hemisection.

Axonal regeneration of different tracts following transplants of human glial restricted progenitors into the injured spinal cord in rats

The goal of this study was to compare the efficacy of human glial restricted progenitors (hGRPs) in promoting axonal growth of different tracts. We examined the potential of hGRPs grafted into a cervical (C4) dorsal column lesion to test sensory axons, and into a C4 hemisection to test motor tracts. The hGRPs, thawed from frozen stocks, were suspended in a PureCol matrix and grafted acutely into a C4 dorsal column or hemisection lesion. Control rats received PureCol only. Five weeks after transplantation, all transplanted cells survived in rats with the dorsal column lesion but only about half of the grafts in the hemisection. In the dorsal column lesion group, few sensory axons grew short distances into the lesion site of control animals. The presence of hGRPs transplants enhanced axonal growth significantly farther into the transplants. In the hemisection group, coerulospinal axons extended similarly into both control and transplant groups with no enhancement by the presence of hGRPs. Rubrospinal axons did not grow into the lesion even in the presence of hGRPs. However, reticulospinal and raphespinal axons grew for a significantly longer distance into the transplants. These results demonstrate the differential capacity of axonal growth/regeneration of the motor and sensory tracts based on their intrinsic abilities as well as their response to the modified environment induced by the hGRPs transplants. We conclude that hGRP transplants can modify the injury site for axon growth of sensory and some motor tracts, and suggest they could be combined with other interventions to restore connectivity.

Manganese-enhanced MRI Offers Correlation with Severity of Spinal Cord Injury in Experimental Models.

Background:Spinal cord injuries (SCI) are clinically challenging, because neural regeneration after cord damage is unknown. In SCI animal models, regeneration is evaluated histologically, requiring animal sacrifice. Noninvasive techniques are needed to detect longitudinal SCI changes.Objective:To compare manganese-enhanced magnetic resonance imaging (MRI [MEMRI]) in hemisection and transection of SCI rat models with diffusion tensor imaging (DTI) and histology.Methods:Rats underwent T9 spinal cord transection (n=6), hemisection (n=6), or laminectomy without SCI (controls, n=6). One-half of each group received lateral ventricle MnCl2 injections 24 hours later. Conventional DTI or T1-weighted MRI was performed 84 hours post-surgery. MEMRI signal intensity ratio above and below the SCI level was calculated. Fractional anisotropy (FA) measurements were taken 1 cm rostral to the SCI. The percentage of FA change was calculated 10 mm rostral to the SCI epicenter, between FA at the dorsal column lesion normalized to a lateral area without FA change. Myelin load (percentage difference) among groups was analyzed by histology.Results:In transection and hemisection groups, mean MEMRI ratios were 0.62 and 0.87, respectively, versus 0.99 in controls (P<0.001 and P<0.001, respectively); mean FA decreases were 67.5% and 40.1%, respectively, compared with a 6.1% increase in controls (P=0.002 and P=0.019, respectively). Mean myelin load decreased by 38.8% (transection) and 51.8% (hemisection) compared to controls (99.1%) (P<0.001 and P<0.001, respectively). Pearson’s correlation coefficients were -0.94 for MEMRI ratio and FA changes and 0.87 for MEMRI and myelin load.Conclusion:MEMERI results correlated to SCI severity measured by FA and myelin load. MEMRI is a useful noninvasive tool to assess neuronal damage after SCI.

Biomechanic study on lumbar zygapophysial joint fusion cage

Objective To investigate biomechanical stability of lumbar zygapophysial joint instrumented with fusion cage.Methods Thirty functional spinal units were divided into control group,lumbar zygapophysial joint fusion cage fixation group,disc semi-resection group,disc semi-resection instrumented with facet screw fixation group and disc semi-resection instrumented with zygapophysial joint fusion cage fixation group.The pull-torsional biomechanical testing machine (MTS 858 System),optical positioning and motion capture system were used to test the angle range of motion (ROM) of functional spinal unit (FSU) under 6 degrees of freedom.Results There was significant difference in the angle ROM in other five degrees of freedom difference except extension orientation between lumbar zygapophysial joint fusion cage fixation group and control group (P ＜ 0.01).After the nucleus hemi-section,the ROM of 6 degrees of freedom in fusion cage fixation group was obviously less than that in the nucleus hemi-section group (P ＜ 0.01),while compared with the facet screw fixation group,there was no significant difference (P ＞ 0.05) except in flexion and extension orientation.Conclusion Lumbar zygapophysial joint fusion cage implantation can strengthen the dynamic stability of functional spinal unit in physiological and nucleus hemi-section conditions. Key words: Lumbar; Zygapophysial joint; Fusion cage ; Biomechanics

Gracile Neurons Contribute to the Maintenance of Neuropathic Pain in Peripheral and Central Neuropathic Models

In the present study, we compared the roles of gracile neurons in mechanically-induced neuropathic pain caused by spinal injury and L5 spinal nerve ligation in rats. Behavioral and electrophysiological methods were used to measure mechanical allodynia in the hindpaws, and excitability of the gracile neurons in the medulla, respectively. In the spinal hemisection and spinal contusion models, mechanical allodynia developed in both hindpaws and lasted over a month. Three weeks following the hemisection, gracile neurons identified as wide-dynamic-range (WDR) and low-threshold (LT) neurons, showed increased neuronal activity to non-noxious mechanical stimuli compared to control groups, whereas the spinal contusion groups did not show evoked activity (*p<0.05). A lesion of the gracile nucleus partially reversed the existing mechanical allodynia in both hindpaws compared to prior to the injury in the hemisection group, whereas the spinal contusion groups did not show significant changes (*p<0.05). In the spinal nerve ligation model, mechanical allodynia developed at the ipsilateral (injured) side of the hindpaw. In addition, WDR neuronal activity at the ipsilateral gracile neurons showed a significant increase with non-noxious mechanical stimuli, whereas the LT neurons did not show significant changes (*p<0.05). Similarly to the hemisection model, a lesion of the gracile nucleus attenuated the mechanical allodynia in spinal nerve ligation models. The present data suggest that gracile neurons contribute to the maintenance of non-noxious mechanically-induced neuropathic pain in both hemisection- and ligation-induced neuropathic pain in rats.

Activation of p-38α MAPK contributes to neuronal hyperexcitability in caudal regions remote from spinal cord injury

In the present study, we examined whether activation of p-38α MAPK modulates mechanical allodynia and neuronal hyperexcitability, and if propentofylline (PPF, a glial modulator) modulates specifically localized activated p-38α MAPK expression in caudal regions remote from a low thoracic hemisection injury in rats. T13 spinal hemisection produces bilateral mechanical allodynia in hindpaws with evoked (in response to mechanical stimuli) neuronal hyperexcitability in lumbar spinal wide dynamic range (WDR) neurons compared to sham controls. The mechanical allodynia and the evoked activity of WDR neurons is attenuated by intrathecal and topical administration of SB203580, an inhibitor of p-38 MAPK activation, dose dependently (⁎ p < 0.05); however, the spontaneous activity showed no significant differences compared to sham controls. After T13 spinal hemisection, significantly increased phosphorylated (activated form) p-38α MAPK expression was present in both superficial and deep dorsal horn neurons as well as in microglia, but not in astrocytes, in the lumbar spinal cord compared to sham controls (⁎ p < 0.05). Intrathecal application of PPF significantly attenuated the expression of phosphorylated p-38α MAPK in superficial dorsal horn neurons (10 mM) and in microglia (1 and 10 mM) in the lumbar spinal cord compared to the hemisection group (⁎ p < 0.05). In conclusion, our present data demonstrate that activated neuronal and microglial, but not astrocytic, p-38α MAPK contributes to the maintenance of neuronal hyperexcitability in caudal regions following spinal cord injury.

Hemisection: one large step toward management of congenitally missing lower second premolars.

When the primary molar cannot be retained in cases of agenesis of the lower second premolar (CML5), there is the possibility of alveolar atrophy, and space closure might have a negative impact on facial fullness. Bearing these in mind, the removal of the distal half of the second primary molar might allow closure in stages. Subsequent removal of the mesial half can be followed by space closure. Using this hemisection approach, space closure can be continued with ease improving treatment results. In order to test the amount of anchorage loss resulting from this approach, the pitchfork analysis was used to compare a group of 23 consecutively treated patients, each treated by hemisection, with two groups of 30 patients, each of whom was treated with the extraction of four premolars. One group had four first premolars removed and the second group had second premolars removed. In order to obtain an untreated "control," these were further compared with the difference between the Bolton templates at ages nine and twelve. The CML5 group was divided into those which had upper teeth extracted in order to facilitate the correction and those who were treated without extraction in the upper. The hemisection groups showed statistically significant diminished distal movement of the upper incisor as well as the upper and lower lip. Lower molar protraction and molar relation was significantly increased. The process facilitates the keeping of upper premolars, which further enhances the facial fullness.

Resection of glial scar following spinal cord injury

While many studies have focused on modulating the immune response and enhancing axonal regeneration after spinal cord injury (SCI), there is limited work being performed on evaluating the role of glial scar in SCI. We sought to evaluate the effects of glial scar resection in contusion models and dorsal hemisection models of SCI. At 1-week postinjury, 2 mm of glial scar was excised from specimens in one of the two groups from each injury model. Functional outcome was measured weekly using the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale along with histologic evaluation of spinal cord tracts to determine axonal regeneration. Within the dorsal hemisection model, there was no significant difference in recovery for animals that underwent glial scar excision versus animals that did not have scar excision (p = 0.61). Animals subjected to the contusion model, however, demonstrated lower BBB scores in the glial resection group during the earlier postoperative periods (< 4 weeks; p < 0.05). Histological analysis revealed no axons within the glial resection contusion model, and moderate axonal growth within the nonresection contusion group and both hemisection groups (p > 0.05 for differences among the three groups). While glial scar may serve to stabilize the preserved axonal tracts and thereby permit modest recovery in a contusion model of SCI, it may be of less importance with a dorsal hemisection model. These experiments highlight that basic biologic processes following SCI may vary tremendously based on the injury mechanism and that the role of glial scar in spinal cord regeneration must be elucidated.

After facial nerve damage, regenerating axons become aberrant throughout the length of the nerve and not only at the site of the lesion: an experimental study

After facial nerve trauma, aberrant regeneration is associated with synkinesis. Animal models of mechanical nerve guides or reparative cell transplants at the site of a lesion have not been shown to improve disorganized regeneration. We examined whether this is because regenerating axons become disorganized throughout the length of the nerve and not only at the site of the lesion. In rats (n = 12), retrograde fluorescent tracer techniques were used to establish that most of the temporal branch fibres were carried in the superior half of the facial nerve trunk. In two further groups of rats (n = 24) a complete proximal facial nerve lesion was made, and the nerve immediately repaired by suture. After 4 weeks, at a second operation, the superior half of the facial nerve trunk was cut, either proximal or distal to the original lesion, and retrograde tracers were applied to distal branches of the nerve. It was possible to localize the points at which regenerating fibres became aberrant in their course by studying the number of labelled motoneurons in the facial nucleus after application of the tracer to the temporal branch of the nerve: this was similar in the distal and proximal hemisection groups, suggesting that aberrant axonal development occurred throughout the length of the nerve. Future strategies aimed at improving the organization of regeneration need to provide guidance cues not only at the site of the lesion as previously thought, but also throughout the length of the nerve.

Regional energy metabolism following short-term neural stem cell transplantation into the injured spinal cord.

Stem cells have been shown to partly restore central nervous system (CNS) function after transplantation into the injured CNS. However, little is known about their influence on acute energy metabolism after spinal cord injury. The present study was designed to analyze regional changes in energy metabolites. Young adult mice were subjected to laminectomy with subsequent hemisection at the L2/3 vertebral level. Immediately thereafter a stable clone of murine neural stem cells (NSCs) was injected into the lesion site. After 4 and 24 h, spinal cords were removed and ATP, glucose, and lactate were analyzed by a bioluminescence approach in serial sections and compared to a laminectomized (intact control), hemisected-only or hemisected vehicle-injected control group. At both time points, ATP content of the hemisected group in the tissue segments adjacent to the lesion was increased when compared to the laminectomized control. At the lesion site ATP content decreased significantly at 24 h in the cell-transplanted group when compared to the laminectomized control group. Glucose content decreased at the lesion site and in segments adjacent to the lesion at both time points and in all experimental groups when compared to the laminectomized control group. Lactate content decreased significantly at 4 h in the caudal segments of the vehicle-injected group and in both adjacent segments of the transplanted group when compared to the laminectomized control. At the lesion site, lactate content decreased significantly at 4 and 24 h in the cell-transplanted group, when compared to the laminectomized control. The area of ATP decline at the lesion site 24 h postinjury was significantly lower in the vehicle control group as compared to the hemisected or transplanted group. The decrease in glucose combined with an increase in ATP in the lesion-adjacent segments may indicate that the tissue responds with an increased use of glucose to support itself with sufficient ATP. The significant decrease in glucose, lactate, and ATP in the cell-transplanted group at 24 h may indicate a high metabolic need of the stem cells. The lower area of ATP decline 24 h after vehicle administration suggests that the vehicle solution washes out toxic mediators, thus ameliorating hemisection-dependent secondary tissue damage.

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 α-CGRP. The increased area and density of GAP-43-IR is consistent with neurite sprouting, and the colocalization with α-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.

Correlation of healing capacity with vascular response in the anterior cruciate and medial collateral ligaments of the rabbit

In clinical terms, functional recovery after anterior cruciate ligament (ACL) injury is generally poorer than after medial collateral ligament (MCL) injury. In experimental studies of injury, the early phases of ligament healing require an augmented blood supply. We hypothesized that the differences in healing properties of the ACL and MCL would be reflected in the magnitude of their vascular responses to partial injury. This study is the first to quantify and define the time course of changes in blood flow and vascular volume following hemisection of the rabbit ACL and MCL. Adult female rabbits were assigned to control, sham operation, ACL hemisection or MCL hemisection groups. Standardized ACL or MCL injuries were surgically induced. About 2, 6 or 16 weeks later, blood flow and vascular volume of the ACL and MCL were measured. The MCL of the rabbit responded to hemisection with a large significant increase in blood flow and a substantial angiogenic response associated with inflammation and scar formation. During subsequent matrix remodelling, blood flow and vascular volume returned towards control values. In contrast, the ACL showed only a 2-fold increase in vascular volume, no increase in blood flow and atrophied after hemisection. The superior capacity of the MCL to increase its blood supply through angiogenesis and increased flow is essential for ligament healing to occur, and may be the major difference in healing potential between the ACL and MCL.

Intrathecal administration of an NMDA or a non-NMDA receptor antagonist reduces mechanical but not thermal allodynia in a rodent model of chronic central pain after spinal cord injury

Spinal cord injuries (SCI) result in a devastating loss of function and chronic central pain syndromes frequently develop in the majority of these patients. The present study uses a rodent spinal hemisection model of SCI in which mechanical and thermal allodynia develops by 24 days after injury. Post-operative paw withdrawal responses to low threshold and high threshold mechanical stimuli compared to pre-operative responses (4.78, 9.96, and 49.9 mN) were increased and were statistically significant ( p<0.05) for both forelimbs and hindlimbs indicating the development of mechanical allodynia. By contrast, post-operatively, the temperature at which paw withdrawal accompanied by paw lick occurred was significantly decreased ( p<0.05), indicating the development of thermal allodynia. The intrathecal application of either D-AP5, a competitive NMDA receptor antagonist, or NBQX-disodium salt, a competitive non-NMDA AMPA/kainate receptor antagonist, alleviated the mechanical allodynia and lowered the threshold of response for the high threshold mechanical stimuli in a dose-dependent manner, and these decreases were statistically significant ( p<0.05). By contrast, neither the D-AP5 nor the NBQX produced a statistically significant change in the thermal allodynia behavior in either forelimbs or hindlimbs in the hemisected group. No significant changes in locomotion scores, and thus no sedation, were demonstrated by the hemisected group for the doses tested. These data support the potential efficacy of competitive excitatory amino acid receptor antagonists in the treatment of chronic central pain, particularly where input from low threshold mechanical afferents trigger the onset of the painful sensation. Furthermore, these data suggest a role for both NMDA and non-NMDA receptors in the development of plastic changes in the spinal cord that provide the underlying mechanisms for central neuropathic pain.

Synaptic plasticity of 5-hydroxytryptamine-immunoreactive terminals in the phrenic nucleus following spinal cord injury: A quantitative electron microscopic analysis

The present study was conducted to examine the plasticity of 5-hydroxytryptamine (5-HT)-immunoreactive terminals in the rat phrenic nucleus following an ipsilateral C2 spinal cord hemisection and 30-day survival period. A retrograde horseradish peroxidase (HRP) labeling technique was used to identify the phrenic motoneurons at the electron microscopic (EM) level. After employing a pre-embedding immunocytochemical technique, the ultrastructural characteristics of 5-HT-immunoreactive terminals were qualitatively and then quantitatively analyzed with a computerized morphometric system before and after injury in separate groups of rats. The results indicated that the majority of the 5-HT-labeled terminals formed axodendritic contacts, but some 5-HT-labeled terminals made axosomatic contacts. 5-HT terminals were associated with either asymmetrical or symmetrical synapses, and some displayed postsynaptic dense bodies. Approximately 2% of the 5-HT terminals had dense-core vesicles. Although the total number of labeled and unlabeled terminals in the phrenic nucleus was reduced after hemisection, the number of 5-HT terminals in the hemisected group was greater than that of the control group. Moreover, the total number and length of asymmetrical and symmetrical synaptic active zones per 5-HT terminal were significantly greater after injury. Finally, the total number of 5-HT terminals with multiple synapses was significantly greater in the hemisected group as compared to controls. It is possible that 5-HT synaptic plasticity may be part of the morphological substrate for the unmasking of the latent crossed phrenic pathway which mediates recovery of the ipsilateral hemidiaphragm paralyzed by C2 spinal cord hemisection.

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, Lissauer's 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.

Ultrastructural quantitative analysis of glutamatergic and GABAergic synaptic terminals in the phrenic nucleus after spinal cord injury.

Quantitative analysis of electron microscopic postembedding immunochemically stained material indicates that 48% of all terminals in the rat phrenic nucleus are glutamatergic and 33% are gamma-aminobutyric acid (GABA)ergic. Three distinct types of glutamatergic terminals were observed in the rat phrenic nucleus: terminals characterized by large, loosely arranged spherical synaptic vesicles (SI) or small, compact spherical synaptic vesicles (Ss) and elongated terminals containing spherical synaptic vesicles with neurofilaments (NFs). All three types of glutamatergic terminals display asymmetrical synaptic membrane densities with postsynaptic dense bodies being present in some of the S-type terminals. The GABAergic immunoreactive terminals in the phrenic nucleus most closely resemble F-type terminals. They are characterized by flattened or pleomorphic synaptic vesicles and symmetric synaptic membrane densities. Among the 48% glutamatergic terminals, 27% are SI, 65% are Ss, and 8% are NFs, respectively. Significantly fewer glutamate, GABA, and unlabeled terminals per unit area are present in the phrenic nucleus 30 days after a C2 spinal cord hemisection as compared to nonhemisected controls. The average number of active zones per terminal, however, is greater in the hemisection group (1.45 +/- 0.03) than in the control group (1.34 +/- 0.03), with the active zones in the glutamate terminals mainly accounting for this difference. Moreover, the length of the active zones in the glutamate terminals was significantly longer in the hemisection group (0.37 +/- 0.013 microns) as compared to the controls (0.24 +/- 0.008 microns). In addition, the mean length of synaptic active zones in GABAergic terminals was also found to be longer in the hemisection group (0.36 +/- 0.022 microns) as compared to controls (0.28 +/- 0.014 microns). Finally, there is also a significantly higher ratio of synaptic active zones to the total number of glutamate-labeled terminals after injury (1.73 +/- 0.08) as compared to controls (1.41 +/- 0.04). The number of double/multiple synapses, the percentages of Sl, Ss, and NFs-type terminals, and the percentages of synaptic active zones contacting either distal dendrites or proximal dendrites/somata do not change significantly 30 days after injury. These results are important for a more complete understanding of the synaptic plasticity that occurs in the phrenic nucleus after spinal cord injury and to show how the plasticity may relate to the unmasking of latent bulbospinal respiratory connections which restore function to the hemidiaphragm paralyzed by an ipsilateral spinal cord hemisection.

In vivo magnetic resonance imaging of fetal cat neural tissue transplants in the adult cat spinal cord

Magnetic resonance (MR) imaging was evaluated for its possible diagnostic application in determining the survival of fetal central nervous system tissue grafts in the injured spinal cord. Hemisection cavities were made at the T11-L1 level of eight adult female cats. Immediately thereafter, several pieces of tissue, either obtained from the fetal cat brain stem on embryonic Day 37 (E-37), from the fetal neocortex on E-37, or from the fetal spinal cord on E-23, were implanted into the cavities made in seven cats. The eighth cat served as a control for the effect of the lesion only. In another group of four animals, a static-load compression injury was made at the L-2 level. Seven weeks later, the lesion was resected in three cases and fragments of either fetal brain-stem or spinal cord tissue were introduced. A small cyst was observed in a fourth cat in the compression injury group and a suspension of dissociated E-23 brain-stem cells was injected into this region of cavitation without disturbing the surrounding leptomeninges. Five months to 2 years posttransplantation, MR imaging was performed with a 2.0-tesla VIS imaging spectrometer by acquiring multislice spin-echo images (TR 1000 msec, TE 30 msec) in both the transverse and sagittal planes. Collectively, these intermediate-weighted images revealed homogeneous, slightly hyperintense signals at the graft site relative to the neighboring host tissue in seven of the 11 graft recipients. Two of the remaining four cats exhibited signals from the graft site that were approximately isointense with the adjacent host spinal cord, and the final two cats and the lesion-only control presented with very hypointense transplant/resection regions. The hyperintense and isointense images were tentatively interpreted as representing viable graft tissue, whereas the hypointense transplant/resection sites were considered to be indicative of a lack of transplant survival or the absence of tissue in the lesion-only control animal. Postmortem gross inspection of fixed specimens and light microscopy verified the MR findings in the control animal in 10 of the 11 graft recipients by showing either transplants and/or cysts corresponding to the MR images obtained. In one cat in the hemisection group, histological analysis revealed a very small piece of graft tissue that was not detected on the MR images. Therefore, it is suggested that within certain spatial- and contrast-resolution limits, MR imaging can reliably detect the presence of transplanted neural tissue in both the hemisected and compression-injured spinal cord of living animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantification of Changes in Serotonin Uptake with Spinal Cord Injury

The high-affinity uptake of [3H]serotonin was studied using spinal cord preparations obtained from normal dogs, dogs made paraplegic by midthoracic transection, and dogs made monoplegic by midthoracic hemisection. Measurements were made at 1, 4, and 8 weeks after surgery. Lumbar spinal cord was removed at the above time periods and a myelin-free synaptosomal fraction was obtained by centrifugation and used for studying high-affinity serotonin uptake. At 1 week, there were no significant differences for Km values (nM) among any of the three groups (138 +/- 20, 128 +/- 22, and 118 +/- 20). The Vmax values (pmol/min/mg of protein) at 1 week for the control group (4.9 +/- 0.6) versus the hemisected group (4.3 +/- 0.7) were not significantly different. The Vmax values at 1 week for the completely transected group (2.3 +/- 0.3; P less than 0.01) were significantly reduced by 53 +/- 7%. At 4 weeks, the Vmax values for the hemisected (1.8 +/- 0.2; P less than 0.05) and transected groups (0.6 +/- 0.2; P less than 0.05) were significantly different as compared to the control group. The mean percent drop in Vmax after hemisection was 56 +/- 4%, close to the predicted value of 50%. The mean percent drop in Vmax after transection was 86 +/- 2%. At 8 weeks the mean Vmax values for the hemisected (1.9 +/- 0.3; P less than 0.05) and transected (0.4 +/- 0.4; P less than 0.05) groups were not significantly different compared to their respective 4-week values. It appears that serotonin uptake can be used to quantitate roughly the degree of spinal cord injury.

Quantification of changes in serotonin uptake with spinal cord injury

The high-affinity uptake of [3H]serotonin was studied using spinal cord preparations obtained from normal dogs, dogs made paraplegic by midthoracic transection, and dogs made monoplegic by midthoracic hemisection. Measurements were made at 1, 4, and 8 weeks after surgery. Lumbar spinal cord was removed at the above time periods and a myelin-free synaptosomal fraction was obtained by centrifugation and used for studying high-affinity serotonin uptake. At 1 week, there were no significant differences for Km values (nM) among any of the three groups (138 +/- 20, 128 +/- 22, and 118 +/- 20). The Vmax values (pmol/min/mg of protein) at 1 week for the control group (4.9 +/- 0.6) versus the hemisected group (4.3 +/- 0.7) were not significantly different. The Vmax values at 1 week for the completely transected group (2.3 +/- 0.3; P less than 0.01) were significantly reduced by 53 +/- 7%. At 4 weeks, the Vmax values for the hemisected (1.8 +/- 0.2; P less than 0.05) and transected groups (0.6 +/- 0.2; P less than 0.05) were significantly different as compared to the control group. The mean percent drop in Vmax after hemisection was 56 +/- 4%, close to the predicted value of 50%. The mean percent drop in Vmax after transection was 86 +/- 2%. At 8 weeks the mean Vmax values for the hemisected (1.9 +/- 0.3; P less than 0.05) and transected (0.4 +/- 0.4; P less than 0.05) groups were not significantly different compared to their respective 4-week values. It appears that serotonin uptake can be used to quantitate roughly the degree of spinal cord injury.