Staining In Spinal Cord Research Articles

Comparison of three techniques for paravertebral brachial plexus blockade in dogs

ObjectiveTo compare success and complication rates, based on staining of nerves and other structures, among three techniques of paravertebral brachial plexus blockade (PBPB) in dogs. Study designProspective randomized design. AnimalsA total of 68 thoracic limbs from 34 dogs. MethodsLimbs were randomly assigned to blind (BL) (n = 24), nerve stimulator-guided (NS) (n = 21) or ultrasound-guided (US) (n = 23) technique. Injections were made with 0.3 mL kg−1 of lidocaine mixed with new methylene blue. Time to perform each block and current used during NS technique were recorded. Dogs were anesthetized during the blocks and euthanized once completed. Dissections were performed to evaluate staining of nerves, spinal cord, mediastinum, pleura and vessels. An anova and Tukey adjustment for time, logistic regression for association between current and nerve staining and a generalized linear mixed model for staining of different structures were used. Significance was considered when p ≤ 0.05. ResultsThe median (range) number of nerves stained was 2 (0–4) with BL, 1 (0–3) with NS and 1 (0–4) with US guided technique. No significant differences in staining of C6, C8 and T1 or other structures were found among techniques. Nerve C7 was more likely to be stained by BL (p = 0.05). Time to perform the blocks was significantly different among techniques, with mean ± SD duration in minutes of 3.6 ± 1.8 with BL, 6.3 ± 2.7 with US and 12.2 ± 5 with NS. The most common complication was staining of the spinal cord (29%, 38% and 39% with BL, NS and US, respectively). ConclusionsSuccess rates were low and complication rates were relatively high, based on staining, with the three techniques. Clinical relevanceThe use of more advanced techniques for PBPB in dogs is not justified according to this study. Clinical significance of the complications encountered in this study should be evaluated.

Recombinant Adeno-Associated Virus-Mediated Global Anterograde Delivery of Glial Cell Line-Derived Neurotrophic Factor to the Spinal Cord: Comparison of Rubrospinal and Corticospinal Tracts in the Rat

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of spinal lower motoneurons. Gene delivery is a promising strategy to deliver therapeutic molecules to these vulnerable cells. However, definition of an optimal route of delivery capable of accessing neurons over a considerable extent of the neuraxis represents a significant logistical problem. Intramuscular vector injections are not ideal as this approach would involve hundreds of injections to completely treat an ALS patient and also would be dependent on retrograde transport of the viral platform of choice. Alternatively, upper motoneurons could deliver trophic factors over considerable distances by anterograde transport after a relatively localized intracerebral injection. To test this approach, the present study was designed to compare the corticospinal (CST) and rubrospinal (RST) tracts for their ability to transport recombinant adeno-associated virus serotype 5 (rAAV5)-derived green fluorescent protein (GFP) or glial cell line-derived neurotrophic factor (GDNF) to the spinal cord. Unilateral injections of rAAV5-GFP into the red nucleus (RN) or motor cortex of normal rats produced GFP-positive fibers in the appropriate descending tracts extending to the lumbar spinal cord. For both tracts, GFP-positive axonal projections into the spinal gray matter were consistently observed. GDNF immunohistochemistry demonstrated that confirmed RN injections resulted in GDNF-positive fibers projecting into spinal gray matter as seen in the GFP group. In contrast, confirmed cortical rAAV5-GDNF injections resulted in less evident staining in spinal cord. Spinal cord GDNF levels were elevated at distances up to 72 mm from the injection sites, and confirmed that RST-related GDNF transport to spinal cord surpassed CST-associated delivery.

Expression of mu-, delta-, and kappa-opioid receptor-like immunoreactivities in rat dorsal root ganglia after carrageenan-induced inflammation

Recently, antisera that recognize unique epitopes of the cloned mu-, delta-, and kappa-opioid, receptors (MOR, DOR, KOR, respectively) have been developed. In the present study MOR-, DOR-, and KOR-like immunoreactivities (LIs) were examined in rat dorsal root ganglia (DRGs, L4-5) after injection of carrageenan (CAR) into the hindpaw. In normal control rats 20.9%, 13.5%, and 9% of the DRG neurons contained MOR-, DOR-, KOR-LI, respectively. A marked upregulation in MOR-LI was observed in DRG neurons 1 and 3 d after inflammation. In contrast, CAR induced a distinct downregulation in DOR- and KOR-LIs. MOR-, DOR-, and KOR-LIs were preferentially localized in small DRG neurons. MOR-LI was often located in patches in the cytoplasm, and in some cells close to the somatic plasmalemma. However, DOR- and KOR-LIs mainly showed a diffuse staining pattern within cytoplasm. Two or even all three receptors could sometimes be found to coexist in DRG neurons. In the spinal cord, these receptors were mainly confined to the superficial dorsal horn, with a somewhat diffuse staining which was strong for MOR-LI, and weak for KOR-LI. DOR-LI had distinctly punctate, varicose distribution. CAG induced-alterations in opioid receptor staining in spinal cord were much less pronounced than those in the DRGs with a small increase in MOR-LI and a slight decrease in DOR-LI ipsilaterally. There was an accumulation of all three types of receptors in the sciatic nerve both proximal and distal to the ligation site as early as 2 hr, indicating both antero- and retrograde transport of multiple opioid receptors. However, DOR-LI accumulation was stronger than that of MOR- and KOR-LIs. Taken together, these results suggest that all three opioid receptors are involved in the response to inflammation and that they may play different roles in this pathological state. The coexistence of MOR, DOR, and KOR in at least some primary sensory neurons provides a substrate for functional interactions between these receptors.

Expression of basic fibroblast growth factor in the nervous system of early avian embryos

Basic fibroblast growth factor (bFGF) promotes the survival of a subpopulation of non-neuronal cells developing from trunk neural crest. It was therefore important to determine whether this factor is present in the nervous system at early developmental stages. Immunocytochemistry using specific polyclonal and monoclonal antibodies was combined with three highly sensitive assays: bFGF-induced proliferation of bovine adrenal cortex-derived capillary endothelial cells (ACE), a radioimmunoassay for bFGF (RIA) and Western blot analysis. bFGF immunoreactivity was localized to the cytoplasm of neuroepithelial cells derived from embryonic day 2 (E2) quail neural tubes and cultured for one day in a chemically defined medium. Specific staining was observed in young sensory neurons in cultures of neural crest clusters as well as in a subpopulation of non-neuronal cells. In cultured E7 dorsal root ganglia, immunostaining was confined to neuronal cell bodies and fibers. In situ, staining of spinal cord and ganglionic neurons appeared on E6 and increased in intensity towards E10. Various mesoderm-derived structures such as the limb buds, the mesenchyme dorsal to the neural tube, the vertebral muscles and cartilage showed specific staining patterns in addition to neural tissue. In agreement with the results of immunocytochemical studies, 1.4ng bFGF per mg protein was detected in spinal cord extracts by RIA as early as E3, its concentration increased to 8.0 ng mg-1 on E5 and then to a maximum of 18.0 ng mg-1 protein on E10, this was followed by a subsequent decrease in concentration in older embryos. On the other hand, high levels of bFGF were present in vertebral tissues from E10 onwards. Extracts of immunopositive tissues were subjected to heparin-Sepharose affinity chromatography and eluted in a stepwise salt gradient. Fractions that eluted from the columns at 2 M NaCl contained a bFGF-like protein as revealed by their ability to stimulate the proliferation of ACE cells and by Western blot analysis. These data demonstrate that bFGF is expressed during early nervous system development in both central and peripheral neurons.