Animal models of spinal cord contusion injuries.

BACKGROUND AND PURPOSE: Traumatic spinal cord injury causes initial mechanical disruption of tissue, leading to a complex secondary sequence of pathophysiologic changes and neurologic impairment. These sequelae depend on the impact force delivered to the spinal cord at the time of injury. Successful clinical evaluation of the efficacy of any therapeutic regimen depends on the reliability and reproducibility of an experimental animal model. We describe a trauma device and the biomechanical parameters required to induce severe or moderate spinal cord contusion injury in cats and rats. METHODS: Recovery after injury was determined by behavioral, electrophysiologic, and histologic evaluations. RESULTS: Behavioral and electrophysiologic tests after injury clearly identified the experimental groups. A stable severe paraplegic state (defined as 6 months for cats and 8 weeks for rats), without evidence of behavioral or electrophysiologic recovery, was induced by a 65-Newton (N) load for cats and a 35-N load for rats. Moderate spinal cord contusion injury, from which cats and rats partially recovered after approximately 3 months and 4 weeks, respectively, was induced by a 45- and 25-N load, respectively. CONCLUSION: Use of these injury conditions provides reliable animal models for studies designed to evaluate potential therapeutic regimens for spinal cord injury.

Preparation of brain-derived neurotrophic factor- and neurotrophin-3-secreting Schwann cells by infection with a retroviral vector.

One reason that the central nervous system of adult mammals does not regenerate after injury is that neurotrophic factors are present only in low concentrations in these tissues. Recent studies have shown that the application of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) acts to encourage the regrowth of motor and sensory fibers after spinal cord injury. Other studies have reported that the regrowth of axons after injury was enhanced by the implantation of Schwann cells, which normally secrete BDNF and NT-3. The purpose of the present study was to genetically modify Schwann cells to secrete increased amounts of BDNF or NT-3 by infection with a retroviral vector. Retroviral vectors were constructed by the ligation of BDNF or NT-3 cDNA to the LXSN vector. Viruses were generated from the plasmid forms of the vectors by transient transfection of PA317 amphotrophic retroviral packaging cells. Viruses were harvested and used to infect the human Schwann cell line designated NF-1T. Northern blot analysis of poly (A+) RNA prepared from Schwann cells that were infected with BDNF- or NT-3-containing virus showed the presence of BDNF or NT-3 mRNA. An enzyme-linked immunosorbent assay (ELISA) for BDNF and NT-3 was performed on media the cells were grown in, and on cellular extracts prepared from the BDNF- and NT-3-infected Schwann cells. The ELISA results demonstrated that the Schwann cells were secreting increased levels of immunologically active BDNF or NT-3. Immunocytochemical staining of these cells revealed the presence of these two neurotrophic factors located in perinuclear granules. These neurotrophic factor-secreting Schwann cells are currently being evaluated for their efficacy in the treatment of spinal cord injury.

Electrophysiological improvement after co-implantation of carbon filaments and fetal tissue in the contused rat spinal cord.

The electrophysiological integrity of the adult rat spinal cord was assessed at the lumbar, lower cervical and cortical levels after the animals sustained a severe contusion injury at the mid-thoracic level (T8) and received either carbon filament cultured with fetal spinal cord tissue implants, fetal tissue implants, or carbon filament implants alone. Somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) were recorded from all animal groups at the end of the 8-week survival period. The results of this study demonstrate that the spinal cord injured animals that received carbon filament cultured with fetal spinal cord tissue implants had the highest degree of electrophysiological recovery, indicating that this combination plays an important role in promoting recovery after injury.

Distribution of alpha 1 subunit isoform of (Na,K)-ATPase in the rat spinal cord.

Three isoforms of the alpha subunit of (Na,K)-ATPase have been identified in the rat central nervous system. Using a probe specific for the alpha 1 isoform, mRNA levels were measured from five sections of the rat spinal cord using slot blot techniques. Assigning a value of 1 to the slope obtained from the cervical section, the upper thoracic section was 2.6 times higher; the midthoracic section was 4.5 times higher; the lower thoracic section was 2.6 times higher; and the lumbar section was 1.7 times higher. The results suggest that alpha 1 isoform mRNA levels are not uniform throughout the spinal cord. In situ hybridization techniques showed that alpha 1 isoform mRNA was diffusely abundant in glial and central canal ependymal cells, while labeled neurons were localized exclusively in laterally located anterior horn neurons in cervical, thoracic, and lumbar segments and in ventromedial neurons in mid-thoracic spinal cord. Also, dorsal root ganglia neurons were extensively labeled at all segments.

Acetylcholine receptor gene expression in skeletal muscle of chronic ethanol-fed rats.

The expression of the neuromuscular acetylcholine receptor (AChR) alpha-subunit gene was evaluated in soleus muscles from an animal model of chronic alcoholism. At 8 weeks of age, test rats were placed on a nutritionally complete liquid diet containing 6.7% ethanol (v/v). Age- and weight-matched control rats were pair-fed an isocaloric liquid diet. After a 16-week diet period, soleus muscles were obtained and total RNA and poly(A)+ RNA were isolated. Muscle RNA levels from ethanol-fed and control rats were comparable. AChR alpha-subunit mRNA was detected by hybridization of muscle poly(A)+ RNA with a 32P-labeled, complementary riboprobe. The steady-state level of AChR alpha-subunit mRNA was reduced by 39% (p less than 0.001) in soleus muscles from the ethanol-fed rats as compared to pair-fed controls. These results suggest that the expression of the AChR alpha-subunit gene is down-regulated after chronic ethanol exposure at a transcriptional or posttranscriptional level.

Temporal relationship between nerve-stump-length-dependent changes in the autophosphorylation of a cyclic AMP-dependent protein kinase and the acetylcholine receptor content in skeletal muscle.

The acetylcholine receptor (AChR) content and the autophosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II (R-II) were evaluated in rats soleus muscles at 24, 30 and 66 hr after surgical denervation by cutting the nerve at a short distance (short-nerve-stump) and at a long distance (long-nerve-stump) from the muscle. AChR content was based on the specific binding of [125I]alpha-bungarotoxin (BUTX); changes in the autophosphorylation of R-II were based upon the predominant in vitro 32P-phosphorylation of a 56-Kd soluble protein in cytosolic fractions of solei. The AChR content and the 32P-autophosphorylation of R-II were increased in samples from short-nerve-stump solei, but not from long-nerve-stump solei, after a denervation-time of 30 hr. This nerve-stump-length dependency indicates that the two denervation effects are not related to the immediate halt of impulse-evoked muscle contractility. Furthermore, the results show that alterations in the 32P-autophosphorylation of R-II occurred before, as well as whenever, increases in the AChR content were found. Speculatively, this temporal relationship may be significant with respect to the potential role of R-II in gene expression.

Role of cholinergic neuromuscular transmission in the neuroregulation of the autophosphorylatable regulatory subunit of cyclic AMP-dependent protein kinase type II and the acetylcholine receptor content in skeletal muscle.

Previously, we found that the in vitro [32P]-autophosphorylation of the regulatory subunit of cyclic AMP-dependent protein kinase type II in rat soleus muscles is subject to a nerve-stump-length-dependent neuroregulation which indicates that this event is dependent upon some neural signal other than the impulse-directed release of acetylcholine. In this investigation, tetrodotoxin and alpha-bungarotoxin were also administered to further differentiate the effect of impulse-directed and spontaneously released acetylcholine upon this event and also upon the appearance of new acetylcholine receptors as measured by the binding of radioiodinated bungarotoxin. A 24 h blockade of cholinergic transmission with either neurotoxin did not change the phosphorylation level of the regulatory subunit, while a significant increase is observed when solei are surgically denervated for this period. The phosphorylation level and also the acetylcholine receptor content were increased only after more prolonged (48-96 h) muscle inactivity was produced with the neurotoxins. However, then their effects may not be solely related to alterations in cholinergic transmission. Taken together, our results do not support a trophic role for spontaneously released acetylcholine with respect to the two neurotrophic events studied.

Decreased acetylcholine receptor content in denervated skeletal muscles infused with nerve extract.

The influence of a concentrated extract of soluble substances from the sciatic nerve upon the acetylcholine receptor (AChR) content in the soleus muscle of adult rats was examined by in vivo infusions. Internal and membrane-inserted AChR were quantitated by the specific binding of 125I-alpha-bungarotoxin (a-BuTX). Interestingly, the nerve extract had no apparent effect unless the soleus muscle was also denervated at the start of the infusion. Then, after 66 hr, substantially less (60-80%) binding of 125I-a-BuTX to AChR was observed compared to denervated solei that did not receive an infusion of nerve extract. However, the concentration of protein in the nerve extract had to exceed 5 mg/ml before this effect was evident. Infusions of phosphate-buffered saline, bovine serum albumin, rat liver extract, or human transferrin had no striking effect upon AChR. The prevention of the characteristic denervation-induced increase in non-junctional AChR by an active component in the nerve extract may be due to a trophic signal for decreased synthesis of AChR, but it is also possible that the degradation of AChR was increased.

Myosin light chain phosphorylation during contraction of turtle heart.

The phosphorylation of myosin P-light chain was determined during the contraction cycle of turtle heart beating 5--8 times/min at 5 degrees C. The hearts were freeze-clamped either in systole or diastole, then homogenized and washed in strong acids in order to completely inhibit myosin light chain kinase and phosphatase and isolate the total P-light chain of the heart. The phospho and dephospho forms of P-light chain were separated by two-dimensional gel electrophoresis and were quantitated by densitometry. Alternatively, the hearts were perfused with 32P and the incorporation of [32P]phosphate into the P-light chain was determined. Both methods demonstrated that in hearts frozen in systole more P-light chain was phosphorylated than in hearts frozen in diastole.

Myosin light chain and membrane protein phosphorylation in various muscles.

Phosphorylation of myosin light chain was compared in various muscles. In 32P-labeled chicken anterior latissimus dorsi and posterior latissimus dorsi the [32P]phosphate content of the 19,000-dalton and 18,000-dalton light chains, respectively, was 1.8-fold higher in muscles tetanized for 5 or 15 s than in the contralateral resting muscles. Similar results had been previously obtained with frog sartorius and semitendinosus muscles. In addition to the radioactive technique, the extent of light chain phosphorylation was also measured by densitometry after separating the phospho and dephospho forms of the light chain with two-dimensional gel electrophoresis. The extent of light chain phosphorylation in tetanized chicken muscles and caffeine-treated frog muscles was not greater than 50%. The extent of phosphorylation of the 19,000-dalton light chain in hearts from several animals (turtle, rat, frog, chicken, dog, and cat) showed major differences. At the extremes it was 76% in turtle and 10% in chicken. the polymorphism of heart light chain was also demonstrated. Not only myosin light chains but also membrane proteins were detected to be phosphorylated. Some of the membrane proteins exhibited increases in phosphorylation during muscle contraction.