Experimental axonal injury triggers interleukin-6 mRNA, protein synthesis and release into cerebrospinal fluid.
Diffuse axonal injury is a frequent pathologic sequel of head trauma, which, despite its devastating consequences for the patients, remains to be fully elucidated. Here we studied the release of interleukin-6 (IL-6) into CSF and serum, as well as the expression of IL-6 messenger ribonucleic acid (mRNA) and protein in a weight drop model of axonal injury in the rat. The IL-6 activity was elevated in CSF within 1 hour and peaked between 2 and 4 hours, reaching maximal values of 82,108 pg/mL, and returned to control values after 24 hours. In serum, the levels of IL-6 remained below increased CSF levels and did not exceed 393 pg/mL. In situ hybridization demonstrated augmented IL-6 mRNA expression in several regions including cortical pyramidal cells, neurons in thalamic nuclei, and macrophages in the basal subarachnoid spaces. A weak constitutive expression of IL-6 protein was shown by immunohistochemical study in control brain. After injury, IL-6 increased at 1 hour and remained elevated through the first 24 hours, returning to normal afterward. Most cells producing IL-6 were cortical, thalamic, and hippocampal neurons as confirmed by staining for the neuronal marker NeuN. These results extend our previous studies showing IL-6 production in the cerebrospinal fluid of patients with severe head trauma and demonstrate that neurons are the main source of IL-6 after experimental axonal injury. (+info)
Experimental induction of retinal ganglion cell death in adult mice.
PURPOSE: Retinal ganglion cells die by apoptosis during development and after trauma such as axonal damage and exposure to excitotoxins. Apoptosis is associated with changes in the expression of genes that regulate this process. The genes that regulate apoptosis in retinal ganglion cells have not been characterized primarily because previous studies have been limited to animal models in which gene function is not easily manipulated. To overcome this limitation, the rate and mechanism of retinal ganglion cell death in mice was characterized using optic nerve crush and intravitreal injections of the glutamate analog N-methyl-D-aspartate (NMDA). METHODS: To expose retinal ganglion cells (RGCs) to excitotoxins, adult CB6F1 mice were injected intravitreally in one eye with NMDA. In an alternative protocol to physically damage the axons in the optic nerve, the nerve was crushed using self-closing fine forceps. Each animal had one or the other procedure carried out on one eye. Loss of RGCs was monitored as a percentage of cells lost relative to the fellow untreated eye. Thy1 expression was examined using in situ hybridization. DNA fragmentation in dying cells was monitored using terminal transferase-dUTP nick-end labeling (TUNEL). RESULTS: RGCs comprise 67.5% +/- 6.5% (mean +/- SD) of cells in the ganglion cell layer (GCL) of control mice based on nuclear morphology and the presence of mRNA for the ganglion cell marker Thy1. One week after optic nerve crush, these cells started to die, progressing to a maximum loss of 57.8% +/- 8.1% of the cells in the GCL by 3 weeks. Cell loss after NMDA injection was dose dependent, with injections of 10 nanomoles having virtually no effect to a maximum loss of 72.5% +/- 12.1% of the cells in the GCL within 6 days after injection of 160 nanomoles NMDA. Cell death exhibited features of apoptosis after both optic nerve crush and NMDA injection, including the formation of pyknotic nuclei and TUNEL staining. CONCLUSIONS: Quantitative RGC death can be induced in mice using two distinct signaling pathways, making it possible to test the roles of genes in this process using transgenic animals. (+info)
Nature of the retrograde signal from injured nerves that induces interleukin-6 mRNA in neurons.
In previous studies, interleukin-6 was shown to be synthesized in approximately one-third of lumbar dorsal root ganglion neurons during the first week after nerve transection. In present studies, interleukin-6 mRNA was found to be induced also in axotomized facial motor neurons and sympathetic neurons. The nature of the signal that induces interleukin-6 mRNA in neurons after nerve injury was analyzed. Blocking of retrograde axonal transport by injection of colchicine into an otherwise normal nerve did not induce interleukin-6 mRNA in primary sensory neurons, but injection of colchicine into the nerve stump prevented induction of interleukin-6 mRNA by nerve transection. Therefore, it was concluded that interleukin-6 is induced by an injury factor arising from the nerve stump rather than by interruption of normal retrograde trophic support from target tissues or distal nerve segments. Next, injection into the nerve of a mast cell degranulating agent was shown to stimulate interleukin-6 mRNA in sensory neurons and systemic administration of mast cell stabilizing agents to mitigate the induction of interleukin-6 mRNA in sensory neurons after nerve injury. These data implicate mast cells as one possible source of the factors that lead to induction of interleukin-6 mRNA after nerve injury. In search of a possible function of inducible interelukin-6, neuronal death after nerve transection was assessed in mice with null deletion of the interleukin-6 gene. Retrograde death of neurons in the fifth lumbar dorsal root ganglion was 45% greater in knockout than in wild-type mice. Thus, endogenous interleukin-6 contributes to the survival of axotomized neurons. (+info)
Acute repair of crushed guinea pig spinal cord by polyethylene glycol.
Acute repair of crushed guinea pig spinal cord by polyethylene glycol. We have studied the responses of adult guinea pig spinal cord white matter to a standardized compression within a sucrose gap recording chamber. This injury eliminated compound action potential (CAP) conduction through the lesion, followed by little or no recovery of conduction by 1 h postinjury. We tested the ability of polyethylene glycol (PEG) to repair the injured axons and restore physiological function. Local application of PEG (1,800 MW, 50% by weight in water) for approximately 2 min restored CAP conduction through the injury as early as 1 min post PEG application. The recovery of the CAP +info)
Recovery of functional response in the nucleus of the solitary tract after peripheral gustatory nerve crush and regeneration.
Single-unit recording and transganglionic tracing techniques were used to assess the properties of, and inputs to, neurons within the rostral nucleus of the solitary tract (NST) after peripheral gustatory nerve injury and regeneration in adult hamsters (Mesocricetus auratus). Tastant-evoked responses were recorded from 43 neurons in animals in which the ipsilateral chorda tympani (CT) nerve was crushed 8 wk earlier (experimental animals) and from 46 neurons in unlesioned control animals. The 89 neurons were separated into three functional clusters named according to the best stimulus for neurons in the cluster: S, sucrose; N, sodium acetate; and H, HCl or KCl. Stimulus-evoked spike rates across all stimuli were 35.4 +/- 4.4% lower in the experimental hamsters. The largest difference in evoked spike rates occurred for neurons in the H cluster, in which the response to KCl also was delayed relative to normal responses. The response of S-cluster units to sucrose and saccharin was also lower in the experimental animals. The mean response rate and the time course of response of neurons in the N cluster did not differ between the two groups. For each cluster, the spontaneous rates and mean response profiles across eight stimuli were very similar in the experimental and control animals, and the breadth of tuning hardly differed. In both groups, Na+ responses in the N cluster were amiloride sensitive, and responses to the water rinse after stimulation with HCl were common in the S cluster. At 8-20 wk after nerve crush, biotinylated dextran tracing of the CT nerve revealed that the regenerated CT fibers did not sprout outside the normal terminal zone in the NST, but the density of the central terminal fibers was 36.9 +/- 6.35% lower than normal. After CT nerve crush and regeneration, the overall reduction in taste-evoked spike rates in NST neurons is likely a consequence of this change in terminal fibers; this in turn likely results from the known reduction in CT fibers regenerating past the crush site. In the face of this reduction, the normal taste-evoked spike rate in N-cluster neurons requires explanation. The observed recovery of normal specificity could be mediated by a restoration of specific connections by primary afferent fibers peripherally and centrally or by central compensatory mechanisms. (+info)
Inactivation of Rho signaling pathway promotes CNS axon regeneration.
Regeneration in the CNS is blocked by many different growth inhibitory proteins. To foster regeneration, we have investigated a strategy to block the neuronal response to growth inhibitory signals. Here, we report that injured axons regrow directly on complex inhibitory substrates when Rho GTPase is inactivated. Treatment of PC12 cells with C3 enzyme to inactivate Rho and transfection with dominant negative Rho allowed neurite growth on inhibitory substrates. Primary retinal neurons treated with C3 extended neurites on myelin-associated glycoprotein and myelin substrates. To explore regeneration in vivo, we crushed optic nerves of adult rat. After C3 treatment, numerous cut axons traversed the lesion to regrow in the distal white matter of the optic nerve. These results indicate that targeting signaling mechanisms converging to Rho stimulates axon regeneration on inhibitory CNS substrates. (+info)
Optic nerve crush: axonal responses in wild-type and bcl-2 transgenic mice.
Retinal ganglion cells of transgenic mice overexpressing the anti-apoptotic protein Bcl-2 in neurons show a dramatic increase of survival rate after axotomy. We used this experimental system to test the regenerative potentials of central neurons after reduction of nonpermissive environmental factors. Survival of retinal ganglion cells 1 month after intracranial crush of the optic nerve was found to be 100% in adult bcl-2 mice and 44% in matched wild-type (wt) mice. In the optic nerve, and particularly at the crush site, fibers regrowing spontaneously or simply sprouting were absent in both wt and bcl-2 mice. We attempted to stimulate regeneration implanting in the crushed nerves hybridoma cells secreting antibodies that neutralize central myelin proteins, shown to inhibit regeneration (IN-1 antibodies) (Caroni and Schwab, 1988). Again, we found that regeneration of fibers beyond the site of crush was virtually absent in the optic nerves of both wt and bcl-2 mice. However, in bcl-2 animals treated with IN-1 antibodies, fibers showed sprouting in the proximity of the hybridoma implant. These results suggest that neurons overexpressing bcl-2 are capable of surviving axotomy and sprout when faced with an environment in which inhibition of regeneration has been reduced. Nevertheless, extensive regeneration does not occur, possibly because other factors act by preventing it. (+info)
A search for the factors responsible for absence of recovery of the normal vascular response to oxytocin following sympathetic nerve injury.
In dogs following crush injury to the lumbar sympathetic trunk, reflex vasoconstriction reappears in 4-6 months but the normal vasodilator response to oxytocin does not return even 12 months after crush. Histochemical examination of the walls of the blood vessels shows that division or crush of the lumbar sympathetic trunk or removal of terminal ganglia leads to decentralization, not denervation of the blood vessels. True denervation follows division or crush of the sciatic and femoral nerves. Following recovery from sciatic or femoral crush the pattern of peripheral innervation appears histochemically normal. However, there is no return of the normal vasodilator response to oxytocin. It is concluded that a normal response to oxytocin does not return even after long-term recovery from sympathetic injury, nor does its effect depend on a normal pattern of peripheral adrenergic innervation, but on an unknown more central activity of the sympathetic nervous system. (+info)