Degeneration and regeneration of motoneuron dendrites after ventral root crush: Computer reconstruction of dendritic fields

Dendritic alteration can be induced by peripheral axotomy of central nervous system neurons. In the following experiment the dendritic profile of rat lamina IX motoneurons in the spinal cord segment under the T2 vertebra after ventral root crush (controls, 14, 30, 60, and 90 days postoperative (DPO), six animals per group) were studied. The spinal cord was impregnated by the Golgi technique, individual dendritic segments were measured from coded slides, the data computerized, and the dendrites reconstructed by computer. The dendritic parameters generated were length of the dendritic segment between branches, total number of branches, number of terminal branches, and serpentine length (total length of entire dendrite). Inter- and intraanimal statistical comparisons were made by ANOVA a priori and the Newman-Keuls test a posteriori. There were statistically significant losses and gains of dendritic branches and numbers of terminal dendrites (lowest, 14 and 60 days; highest, 30 and 90 days). The serpentine length of the dendrites showed the same statistically significant oscillations. There were statistically significant differences in average dendritic parameters (calculated from distance along dendritic segments) after ventral root crush. However, 17% of the neurons at 14 days, 100% at 30 days, 33% at 60 days, and 17% at 90 days differed from control values in serpentine length and at 14 DPO, 50%; 30 DPO, 50%; 60 DPO, 67%; and 90 DPO, 70% of the neurons had numbers of dendritic segments different from control. These data show that there is a cyclic degeneration and regeneration of motoneuron dendrites after ventral root crush for at least 90 postoperative days.     

Patterns of reafferentation in rat nucleus gracilis after thoracic dorsal column lesions

The effect of a bilateral, dorsal column lesion (T12) on the frequency of boutons on nucleus gracilis (NG) neurons was studied. Rats were killed 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days postoperative (DPO), and bouton counts were taken on the soma, or for 5- and 10-μm distances on the proximal dendrite branchings from the soma, in the rostral (level of area postrema), middle, and caudal NG. Significant changes in boutons on the soma occurred mainly during the first week postlesion, in the caudal and middle NG. Only at 7 DPO were the counts significantly less than normal; they increased dramatically at 90 DPO. Bouton frequency correlated significantly during 120 days postlesion between the caudal and middle NG. These data are consistent with previous reports on the distribution in the NG of hind limb afferent fibers coursing in the thoracic dorsal columns; the increase in boutons at 90 DPO is supported by data in other systems. All NG levels showed changes in bouton counts on the proximal dendrite: (i) during the first week postlesion, bouton frequency decreased at 1 and 3 DPO, and increased at 2 and 7 DPO, significantly; (ii) at 7 and 2 DPO the counts were significantly higher than at most longer postlesion times; (iii) only bouton counts at 5 μm on the proximal dendrite (rostral NG) correlated significantly, and negatively, with counts on the soma during 120 days postlesion. These data emphasize the dynamic changes in afferentation of NG neurons during the first postlesion week, which parallel the progressive stages of bouton degeneration and phagocytosis as reported in several sensory systems.     

Blood-brain barrier disturbance and reconstitution after spinal cord hemisection in the rat

Electrophysiologic characteristics and ultrastructural features of single muscle fibers of the dystrophic mouse (C57BL/6J-dy^2J) at various stages of disease were investigated and comparison with those of normal muscle. Resting membrane potentials of the extensor digitorum longus muscle fibers from both normal and dystrophic young mice (to 3 weeks) were between ?90 and ?40 mV. With advancing ages to 7 to 9 weeks, the membrane potentials of normal fibers increased and tended to be within a narrow range around the mean value (?74 mV), whereas those of dystrophic fibers remained at the same value as at 3 weeks (?63 mV), with large deviations. The twitch tension of single fibers induced by direct stimulation was drastically reduced in dystrophic muscle, approximately from one-half to one-third of what it had been in normal fibers. Three-dimensional structures of the T-system in normal muscle observed under a conventional electron microscope revealed the formation of a dense network of tubules along the A-I boundaries of the myofibrils. In dystrophic muscle, the network was poorly developed and the tubules looked disrupted, although the arrangement of myofilaments was preserved relatively well even in the advanced stages. It seems likely that the drastic reduction of contractile force in dystrophic muscle is due to the defect of the internal membrane system, rather than the degeneration of contractile elements.     

Astrocytes secrete basal lamina after hemisection of rat spinal cord

Basal lamina is reconstructed over the lesioned surface of the spinal cord. The following experiment (90 rats) studies the ultrastructure of the formation of this membrane and the immunohistochemistry of laminin production (a major secreted component of basal lamina). After hemisection of the spinal cord at T6 animals were prepared for electron microscopy or antilaminin-biotin-avidin-peroxidase incubation. Three-5 days posthemisection, antilaminin reaction product was observed in astrocytes and their processes which faced the lesion, endothelia of blood vessels or pia. Ultrastructurally (3 days), basal lamina was polymerizing as small projections on the surface of astrocytic membranes facing the lesion, endothelia or pia. By 5 days the basal lamina was a single membrane, folded multiple sheets or in swirls. At 6-10 days the antilaminin reaction and the basal lamina (except for duplications) did not differ from normal. Reactive astrocytes secrete laminin for at least 3-5 days after hemisection and form basal lamina on the lesioned surface of the spinal cord after spinal cord hemisection.     

Amino acid uptake in the spinal cord and brain of the rat after short-term spinal hemisection

The uptake of [³H]lysine was used to study the amino acid incorporation into trichloracetic acid-precipitable protein and soluble fractions of the spinal cord of rats subjected to a left hemisection at T2. Groups of animals were studied 3, 6, and 12h and 1, 3, 7, and 14 days after hemisection. Each group consisted of two sham-operated, and five spinal hemisected animals; five intact normal animals were also studied. Samples were taken from left and right somatomotor and occipital cortex and from both sides of the spinal cord as far as 16 mm rostral and caudal to the site of hemisection. Amino acid uptake into the protein and acid-soluble fractions exhibited significant alteration in [³H]lysine incorporation with time in both sham-operated and operated animals. Radioactivity of the trichloracetic acid-soluble fraction was elevated above normal in both groups at 3 h postoperative in the spinal cord and by 6 h in the brain. However, overall increases in acid-precipitable protein radio-activity did not appear until 6 h after hemisection and 6 h after the sham operation. These increases appeared to be associated with operative stress and/or trauma. At the site of lesion, local elevations in the soluble-fraction radioactivity were noted as early as 3 h after hemisection and as far as 4 mm rostral. Elevations in the acid-precipitable protein at the lesion site were not seen until 3 days postoperative and were generally restricted to the left (hemisected) side of the spinal cord. The local increases were attributed to increases in neuronal, neuroglial, and infiltrating cell metabolism possibly associated with regenerative changes.     

Staircase but not posttetanic potentiation in rat muscle after spinal cord hemisection

Spinal cord hemisection (SCH) results in atrophy of skeletal muscle and altered contractile properties. In this study our purpose was to assess staircase and posttetanic potentiation in the rat gastrocnemius muscle in situ, 1 week after SCH. Tetanic force was reduced by SCH, but twitch amplitude was not. The time course and magnitude of staircase during stimulation at 5 HZ (for 21 s) was similar in the control, sham-operated, and SCH groups. However, posttetanic potentiation observed after 100-, 500-, and 1000-ms tetanic (200 HZ) contractions was absent or drastically reduced after SCH. Twitch force increased 44+/-8.7%, 47+/-7.4%, and 15+/-2.8% for the control, sham, and SCH groups, respectively, after the 1000-ms tetanic contraction. After the 1000-ms tetanic contraction, twitch active force decreased in all groups and was significantly reduced at 5 min relative to pretetanic twitch. In the control and sham groups, but not SCH, the active force recovered to the pretetanic level by 15 min. Resting regulatory light chain (RLC) phosphorylation was 15.4+/-2.5% and 10.97+/-3.3% for the control and SCH groups, respectively. After the 1-s tetanic contraction, values were 41.6+/-2.8% and 9.3+/-2.9%, respectively. The potentiation observed in the SCH animals with 1000-ms contraction apparently occurred without increases in RLC phosphorylation. One week after SCH there were clear changes in the contractile properties typically associated with prior activation. It is concluded that activity-dependent potentiation can occur by a mechanism that is independent of RLC phosphorylation, and accelerated fatigue can mask the potentiating effects of prior activity.     

Changes in truncated trkB and p75 receptor expression in the rat spinal cord following spinal cord hemisection and spinal cord hemisection plus neurotrophin treatment

Although numerous studies have examined the effects of neurotrophin treatment following spinal cord injury, few have examined the changes that occur in the neurotrophin receptors following either such damage or neurotrophin treatment. To determine what changes occur in neurotrophin receptor expression following spinal cord damage, adult rats received a midthoracic spinal cord hemisection alone or in combination with intrathecal application of brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3). Using immunohistochemical and in situ hybridization techniques, p75, trkA, trkB, and trkC receptor expression was examined throughout the spinal cord. Results showed that trkA, full-length trkB, and trkC receptors were not present in the lesion site but had a normal expression pattern in uninjured parts of the spinal cord. In contrast, p75 receptor expression occurred on Schwann cells throughout the lesion site. BDNF and NT-3 (but not saline) applied to the lesion site increased this expression. In addition, the truncated trkB receptor was expressed in the border between the lesion and intact spinal cord. Truncated trkB receptor expression was also increased throughout the white matter ipsilateral to the lesion and BDNF (but not NT-3 or saline) prevented this increase. The study is the first to show changes in truncated trkB receptor expression that extend beyond the site of a spinal cord lesion and is one of the first to show that BDNF and NT-3 affect Schwann cells and/or p75 expression following spinal cord damage. These results indicate that changes in neurotrophin receptor expression following spinal cord injury could influence the availability of neurotrophins at the lesion site. In addition, neurotrophins may affect their own availability to damaged neurons by altering the expression of the p75 and truncated trkB receptor.     

Patterns of afferentation in rat ventroposterolateral nucleus after thoracic dorsal column lesions

The secondary transneuronal effect of bilateral, dorsal column lesion (T12) on the frequency of boutons on cells in the lateral ventroposterolateral nucleus (VPL) was studied. Rats were killed 1, 2, 3, 7, 14, 30, 45, 60, 90, or 120 days postoperative (DPO), and bouton counts (Rasmussen stain) were made on the soma, or along 5- and 10-μm segments of the proximal dendrite branching from soma, in the rostral two-thirds of the VPL. The soma diameter also was measured of those neurons chosen for bouton counts on the circumference of the soma. Significant, increased afferentiation along a 5-μm segment of the proximal dendrite occurred during the first 3 days postlesion compared with longer survival times. Along the 10-μm segment, as at 5 μm on the proximal dendrite, bouton counts at 2 DPO were significantly higher than at 30 and 120 DPO. This rapid change in afferentation during the first week postlesion mirrors similar effects reported in the nucleus gracilis of these same cases. Bouton counts along the 5-μm segment of the proximal dendrite decreased significantly to less than normal at 30 and 120 DPO, indicating the nonstable and protracted character of afferentation after the distal cord lesion. A significant, positive correlation was obtained between boutons on the soma and proximal dendrite (5 μm segment) in cases with lesions, suggesting parallel innervation patterns for these adjacent neuronal components during 120 postoperative days. There was no significant change in the bouton counts or diameter of the soma during postoperative days. These results suggest that lesion of the thoracic dorsal column may result in subsequent rapid bouton patterning on the lateral VPL neurons, presumably mediated by their medial lemniscal afferent fibers. The postlesion source(s) of afferentation remains an intriguing, and as yet unanswered, question.     

Enzyme histochemical changes after transection or hemisection of the spinal cord of the rat

The spinal cords of 84 young adult female rats were transected or hemisected at T7 to T8 and the animals autopsied at intervals from 6 h to 14 months postoperatively. Frozen sections of the unfixed spinal cord on either side of the lesion were prepared for enzyme histochemistry, immunocytochemistry, and histology. The most striking enzymatic alterations and their physiological implications were: (i) (Na⁺---K⁺)-activated ATPase activity decreased in axons of the gray and white matter within 6 h after spinal transection and did not return subsequently, whereas the decrease in activity that occurred contralateral to a hemisection was transient. The decreased activity occurred so promptly as to suggest possible roles in the genesis of the initial flaccid paralysis (spinal shock) in the spinal animal and in the temporary paraplegia seen after subtotal spinal injury. (ii) During the first week postoperatively, many axons in the white matter developed large swellings or small varicosities that reacted strongly only for enzymes normally present in the neuronal perikaryon (e.g., AChE, acid phosphatase, NADH-diaphorase, and G6PDH). This histopathological reaction gradually spread rostrally and caudally from the site of injury, but it disappeared as axonal degeneration supervened. (iii) Within 7 days after spinal transection, many neuronal perikarya were chromatolytic and exhibited decreased AChE activity but normal or increased NADH-diaphorase activity. This response is similar to that seen in the cell bodies of regenerating peripheral axons where anabolic processes are favored over neurotransmission-related functions. (iv) Increased cellularity of the spinal parenchyma adjacent to the lesion resulted largely from the proliferation and hypertrophy of astrocytes. These hypertrophied cells, whose identity was confirmed by GFAP immunocytochemistry, reacted with marked intensity for NADH-diaphorase, G6PDH, and Gly3PDH. Such enzyme changes, characteristic of increased protein turnover, indicate that experimental attempts to control gliosis (e.g., by reducing protein turnover or by other means) could be effectively monitored by enzyme histochemistry.     

NGF levels decrease in the spinal cord and dorsal root ganglion after spinal hemisection

To examine changes in nerve growth factor (NGF) levels in spinal cord and dorsal root ganglia (DRG) after spinal injury, male Sprague-Dawley rats weighing 150-175 g were given spinal hemisections. NGF content was measured at various post-surgical times and compared with naive controls (n = 4 per time point) in the spinal cord, DRG and blood serum by ELISA techniques (Promega). Levels of NGF in the blood serum were significantly increased 8-fold at 48h but were significantly decreased in the spinal cord and DRG by 2- to 4-fold until 7 days postsurgery (ANOVA, p < 0.05). Contrary to accepted dogma, spinal injury results in decreased levels of NGF in the spinal cord and DRG following spinal injury.     

Recovery of locomotor function after hemisection of the spinal cord in cats

Cats were subjected to high lumbar hemisection of the spinal cord, on the right side. The initial paralysis of the right hindlimb became rapidly attenuated, and they walked again in one week or less after surgery. Minor residual deficits in gait remained, that may be permanent. Electrical stimulation of the bulbar reticulospinal formation showed residual crossed connections reaching the right lumbosacral cord via the left hemicord. Recovery from Brown-Séquard's syndrome may be primarily due to the survival of low crossing descending projections to the spinal cord.     

Temporal plasticity of dorsal horn somatosensory neurons after acute and chronic spinal cord hemisection in rat

Unilateral T13 hemisection of the rat spinal cord produces a model of chronic spinal cord injury (SCI) that is characterized by bilateral hyperexcitability of lumbar dorsal horn neurons, and behavioral signs of central pain. While we have demonstrated that responsiveness of multireceptive (MR) dorsal horn neurons is dramatically increased at 28 days after injury, the effects of acute hemisection are unknown and predicted to be different than observed chronically. In the present study, the consequences of T13 hemisection are examined acutely at 45 min in MR neurons both ipsilateral and contralateral to the site of injury, and compared to the same class of cells at 28 days after injury (n=20 cells total per group: 2–3 cells/side of the cord from n=5 animals). Acutely, ipsilateral to the hemisection, both spontaneous and evoked activity of MR neurons were significantly increased, whereas contralaterally, only evoked activity was significantly increased. In animals 28 days after hemisection, spontaneous activity of MR neurons was comparable to intact levels ipsilaterally, and cells exhibited hyperexcitability to evoked stimuli bilaterally. Expansion of cutaneous receptive fields was observed only in hindpaws ipsilateral to the lesion, acutely. These results demonstrate dynamic plasticity in properties of dorsal horn somatosensory neurons after SCI.     

Effect of puromycin administration on presynaptic bouton regeneration after hemisection of rat spinal cord.

The effect of puromycin on synaptic renewal on spinal neurons 1 to 1.5 mm rostral to the site of a T1-T2 interface after hemisection was studied. Ninety-five Long-Evans rats were utilized (five animals per group) 10, 20, 30, 45, 60, and 90 days posthemisection. The groups consisted of normal animals, those with spinal cord hemisection alone, those with Gelfoam sponge saturated with 0.9% saline placed in the lesion, or those with a Gelfoam sponge saturated with 1.0 mm puromycin dihydrochloride in 0.9% saline placed in the lesion (at surgery). The spinal cords were processed for the light microscopic impregnation of presynaptic boutons. Counts of boutons (lamina IV and IX motoneurons) were made from coded material and analyzed statistically. After spinal cord hemisection alone, there was a cyclic significant renewal of presynaptic boutons on the somata of lamina IV and IX neurons with decreases (P < 0.05) at 10 to 20 days, increases (P < 0.05) at 30 days, and spontaneous loss (P < 0.05) at 45 days. This small number of boutons was retained at 60 days postoperative (DPO) and remains at this value at 90 days on lamina IV neurons or increases on lamina IX neurons. Saline-puromycin-Gelfoam implantation resulted in a significant bouton decrease (P < 0.05) on lamina IX neurons at only 45 days postoperative and no differences from normal on lamina IV neurons. These data show a “protective” effect that stabilized presynaptic boutons on lamina IV neurons for 90 days and on lamina IX neurons for at least 30 days.     

Erythropoietin and carbamylated erythropoietin are neuroprotective following spinal cord hemisection in the rat

The cytokine erythropoietin (EPO) has been shown to be neuroprotective in a variety of models of central and peripheral nervous system injury. Derivatives of EPO that lack its erythropoietic effects have recently been developed, and the initial reports suggest that they have a neuroprotective potential comparable to that of EPO. One such derivative is carbamylated EPO (CEPO). In the current study we compared the effects of treatment with EPO and CEPO on some of the early neurodegenerative events that occur following spinal cord injury (SCI) induced by hemisection. Adult male Wistar rats received a unilateral hemisection of the spinal cord. Thirty minutes and 24 h following injury, animals received an intraperitoneal injection of saline, EPO (40 microg/kg) or CEPO (40 microg/kg). Results indicated that 3 days post-injury, both CEPO and EPO decreased to a similar extent the size of the lesion compared with control animals. Both compounds also decreased the number of terminal transferase-mediated dUTP nick-end labelling (TUNEL)-labelled apopotic nuclei around the lesion site, as well as the number of axons expressing the injury marker beta-amyloid precursor protein. EPO and CEPO also increased Schwann cell infiltration into the lesion site, although neither compound had any effect on macrophage infiltration either within the lesion site itself or in the surrounding intact tissue. In addition, immunohistochemistry showed an increased expression of both the EPO receptor and the beta common receptor subunit, the components of the receptor complex proposed to mediate the neuroprotective effects of EPO and CEPO in neurons near the site of the injury. The results show that not only does CEPO have an efficacy comparable to that of EPO in its neuroprotective potential following injury, but also that changes in the receptors for these compounds following SCI may underlie their neuroprotective efficacy.     

Neuroprotective and neurodestructive functions of nitric oxide after spinal cord hemisection

Nitric oxide (NO) may subserve different functions in different central neurons subjected to axotomy. The difference may depend on whether the neurons basally express neuronal nitric oxide synthase (nNOS), a biosynthetic enzyme of NO. This is supported by our previous finding that suggests the differential role of NO in neurons of nucleus dorsalis (ND) and red nucleus (RN) which have different basal expression of nNOS. This study aimed to establish firmly the functions of NO, as revealed by nNOS immunoreactivity and nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, by the administration of endogenous NO donor, l-arginine (l-arg), and NOS inhibitor, l-N(G)-nitroarginine methyl ester (l-NAME). To relate the role of NO to glutamate receptors (GluR), the distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) in the two nuclei were revealed by immunohistochemical techniques. nNOS immunoreactivity was void in ND neurons, but expressed weakly in the RN normally. It was induced in ipsilateral ND neurons and upregulated on both sides of RN after spinal cord hemisection. Neuronal loss in the ipsilateral ND was augmented by l-arg, but reduced by l-NAME. In the contralateral RN, l-arg attenuated neuronal loss. NMDAR1 was present in most neurons in ND. After axotomy, some NMDAR1 immunoreactive neurons of the ipsilateral ND were induced to express NOS, whereas RN neurons showed strong staining for NMDAR1 and all the AMPA subunits. Most of the NOS-positive neurons in the RN were coexistent with GluR2 in normal rats and those subjected to axotomy. The present data demonstrated that NO exerted neurodestructive function in the non-NOS-containing ND neurons characterized by NMDAR as the predominant glutamate receptor. NO might be beneficial to the NOS-containing RN neurons. This could be attributed to the presence of GluR2. Possible diverse synthesizing pathways of NO in two different central nuclei were suggested from the observation that NOS was colocalized with NADPH-d in ND neurons, but not in RN neurons.     

Theophylline treatment improves mitochondrial function after upper cervical spinal cord hemisection

The importance of mitochondria in spinal cord injury has mainly been attributed to their participation in apoptosis at the site of injury. But another aspect of mitochondrial function is the generation of more than 90% of cellular energy in the form of ATP, mediated by the oxidative phosphorylation (OxPhos) process. Cytochrome c oxidase (CcO) is a central OxPhos component and changes in its activity reflect changes in energy demand. A recent study suggests that respiratory muscle function in chronic obstructive pulmonary disease (COPD) patients is compromised via alterations in mitochondrial function. In an animal model of cervical spinal cord hemisection (C2HS) respiratory dysfunction, we have shown that theophylline improves respiratory function. In the present study, we tested the hypothesis that theophylline improves respiratory function at the cellular level via improved mitochondrial function in the C2HS model. We demonstrate that CcO activity was significantly (33%) increased in the spinal cord adjacent to the site of injury (C3-C5), and that administration of theophylline (20 mg/kg 3x daily orally) after C2HS leads to an even more pronounced increase in CcO activity of 62% compared to sham-operated animals. These results are paralleled by a significant increase in cellular ATP levels (51% in the hemidiaphragm ipsilateral to the hemisection). We conclude that C2HS increases energy demand and activates mitochondrial respiration, and that theophylline treatment improves energy levels through activation of the mitochondrial OxPhos process to provide energy for tissue repair and functional recovery after paralysis in the C2HS model.