Horseradish peroxidase retrograde labeling of primary sensory neurons: a comparison of four intraspinal application methods.

The purpose of this study was to optimize the methods of retrograde labeling of sensory neurons in demonstrating the continuity of post-ganglionic primary sensory axons. This was accomplished by comparing four different methods of horseradish peroxidase (HRP) application into the lower thoracic spinal cord of adult rats (level T11). HRP application with a piece of Gelfoam via a dorsal myelotomy (group 1, n = 8), stereotactic injections with a 0.72-mm tip diameter needle (group 2, n = 8), with a 0.24-mm tip needle (group 3, n = 8), and with a 0.08-mm tip glass micropipette (group 4, n = 5). Histological examination of the application site showed that the extent of spinal cord injury was directly proportional to the diameter of the needle tip. The mean number of dorsal root ganglia (DRG) sensory neurons retrogradely stained by HRP differed among the four experimental groups: 77 +/- 45 (SEM) per DRG in group 1, 106 +/- 24 in group 2, 652 +/- 90 in group 3, and 238 +/- 60 in group 4. A significant difference was found between group 3 and the other ones (P < 0.05). Intraspinal injection of HRP with a fine needle (0.24-mm tip diameter) using a stereotactic approach can achieve effective and reliable retrograde labeling of primary sensory neurons. This reproducible method may be useful in studies dealing with regeneration of post-ganglionic primary sensory axons.

[Horseradish peroxidase retrograde labeling of primary sensory axons in rats: a comparison between three different intraspinal injections methods]. / Marquage rétrograde des axones sensitifs primaires par la peroxydase du raifort chez le rat: mise au point de la technique d'injection intramédullaire.

STUDY AIM: In order to improve the results of intraspinal retrograde labeling of post-ganglionic primary sensory axons by horseradish peroxidase (HRP), the authors compared three different intraspinal injection methods of this tracer into the inferior thoracic spinal cord in the rat. MATERIAL AND METHOD: 'Open field' method (group 1, N = 8); stereotactic injection, needle tip diameter = 0.72 mm (group 2, N = 8); stereotactic injection, needle tip diameter = 0.24 mm (group 3, N = 8). Histological features of the spinal injection site showed that tissue damages due to injection was more extensive and deeper than expected. HRP transported in retrograde fashion from injection site to sensory body cells located in dorsal root ganglia (DRG) was revealed by the Mesulam histochemical technique. RESULTS: The mean number of labeled neurons per DRG was 652 in group 3, 116 in group 2, and 77 in group 1. Differences were statistically significant, especially between groups 1 and 3 (P = 4.10(-16)) and groups 2 and 3 (P = 2.10(-17)). CONCLUSION: Retrograde labeling of primary sensory axons by HRP (or another axonal tracer) with fine needle stereotactic intraspinal injection may represent an alternative to anterograde labeling. This reliable and reproducible method may be useful in studies dealing with regeneration of post-ganglionic primary sensory axons.

Expression of cytokines by human astrocytomas following stimulation by C3a and C5a anaphylatoxins: specific increase in interleukin-6 mRNA expression.

C3a and C5a anaphylatoxins are two proinflammatory peptides generated during complement activation that act through distinct Gi protein-coupled receptors named C3aR and C5aR, respectively. We have demonstrated previously that human astrocytes expressed C3aR and C5aR constitutively and were able to produce a functional complement. In this study, we examined the effect of an anaphylatoxin stimulation on cytokine expression by human astrocyte cell lines. Interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA expression was studied by quantitative RT-PCR. Whereas IL-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta mRNA levels remained unchanged, stimulation of astrocytoma cells (T98G, CB193, U118MG) by C3a, C5a, and peptidic C3aR and C5aR agonists induced an increase in the IL-6 mRNA level. The amount of IL-6 was markedly increased at 3 and 6 h and returned to the basal level at 9 h of stimulation. This response was specific, because pretreatment of cells with pertussis toxin or with polyclonal anti-C3aR or anti-C5aR antibodies completely blocked the IL-6 mRNA increase. The IL-6 response was also investigated at the protein level, but IL-6 protein was detected neither in cell lysates nor in supernatants of stimulated cells. The anaphylatoxin-mediated transcriptional activation of IL-6 gene suggests that C3a and C5a could play a role in priming glial cells during the inflammatory process in the brain.

Axonal regrowth through a collagen guidance channel bridging spinal cord to the avulsed C6 roots: functional recovery in primates with brachial plexus injury.

Intraspinal implantation of a collagen guidance channel (CGC) to promote axon regeneration was investigated in marmosets with brachial plexus injury. After avulsion of the right C5, C6 and C7 spinal roots, a CGC containing (group B) or not (group A) a nerve segment, or a nerve graft (group C), was ventro-laterally implanted into the cord to bridge the ventral horn and the avulsed C6 roots. No spinal cord dysfunction was observed following surgery. Two months later, the postoperative flaccid paralysis of the lesioned arm improved. In five months, a normal electromyogram of the affected biceps muscle was recorded in all repaired animals. Motor evoked potentials were obtained with a mean amplitude of 13.37 +/- 13.66 microV in group A, 13.21 +/- 5.16 microV in group B and 37.14 +/- 35.16 microV in group C. The force of biceps muscle contraction was 27.33 +/- 20.03 g (group A), 24.33 +/- 17.03 g (group B) and 37.38 +/- 21.70 g (group C). Retrograde tracing by horseradish peroxidase showed labelled motoneurons ipsilaterally located in the C5 and C6 ventral horn, nearby the implantation site. The mean labelled neurons was 32.33 +/- 21.13, 219.33 +/- 176.29 and 64.33 +/- 23.54 in group A, B and C respectively. Histological analysis presented numerous myelinated and unmyelinated regenerating axons in the implant of these animals. Statistical analysis did not show significant difference among the three repaired groups. Our results indicate that spinal neurons can regenerate through a CGC to avulsed nerve roots and induce motor recovery in primates.

Expression of clusterin and C4 mRNA during rat peripheral nerve regeneration.

The complement system (C) is a major piece of the inflammatory processes triggered after tissue injury. Since implication of the C has been demonstrated during neurodegeneration and Wallerian degeneration, without being clearly explained, we investigated the expression of C4 and clusterin mRNA, at the lesion site, after rat sciatic nerve crush injury. This pilot study was then realized over 28 days during peripheral nerve regeneration. We determined mRNA expression levels in naive control animals (N) and 2, 7, 14 and 28 days (D) after crush experiment, by using semi-quantitative RT-PCR. We observed a basic constitutive expression of both mRNAs in group N. Clusterin mRNA level increased between D2 and D7 to reach 2.5-fold the basic level of expression (N) at D7 and D14, and slightly decreased until D28. C4 mRNA underwent a rapid and marked increase and represented 2-3-fold the N level from D2 to D14, then it decreased until D28 to return to the basic level of expression (N). These preliminary data exhibit very interesting individual variations in mRNA expression and show that a peripheral nerve trauma can stimulate the expression of C4 and clusterin mRNA at the lesion site.