Burn Education Awareness Recognition and Support (BEARS): a community-based juvenile firesetters assessment and treatment program.

In response to the continued staggering statistics of fires set by juveniles and the devastating personal and property costs that are associated with these fires, the Burn and Shock Trauma Institute of Loyola University Medical Center, in collaboration with the State Fire Marshal's Office; the Illinois Fire Safety Alliance; and representatives from the firefighting community, law enforcement, emergency medicine and mental health, came together to create the Burn Education Awareness Recognition and Support Program. Through financial grant support from the International Association of Firefighters, the Illinois Fire Safety Alliance, and other private donations, the Burn Education Awareness Recognition and Support Program is able to provide a free resource to anyone who is concerned about a child playing with fire. Specially trained firefighters assess each child using the tool developed by the Federal Emergency Management Agency. In 2002, we assessed 42 children; 29 of those children were referred through the courts. So far, none of the children treated in our program have returned to fire-setting behaviors.

Effects of interleukin-10 (IL-10) on pain behavior and gene expression following excitotoxic spinal cord injury in the rat.

Intraspinal injection of quisqualic acid (QUIS) produces excitotoxic injury with pathophysiological characteristics similar to those associated with ischemic and traumatic spinal cord injury (SCI). Responses to QUIS-induced injury include an inflammatory component, as well as the development of spontaneous and evoked pain behaviors. We hypothesized that QUIS-induced inflammation and subsequent gene expression contribute to the development and progression of pain-related behaviors and that blockade of inflammation-related gene expression leads to the amelioration of these behaviors. Using the QUIS model of spinal cord injury, we examined whether interleukin-10 (IL-10), a potent anti-inflammatory cytokine, is able to reduce mRNA levels of inflammatory and cell death-related genes leading to a reduction of pain behaviors. The results demonstrate that animals receiving systemic injection of IL-10, 30 minutes following QUIS-induced SCI, showed a significant delay in the onset of excessive grooming behavior, a significant reduction in grooming severity, and a significant reduction in the longitudinal extent of a pattern of neuronal loss within the spinal cord characterized as "grooming-type damage." QUIS injections also resulted in an increase in mRNA levels of interleukin-1 beta (IL-1 beta), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), CD95 ligand (CD95-L, also called FAS-L/APO-1L), and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Results of QUIS injury plus IL-10 treatment resulted in a significant downregulation of IL1-beta and iNOS mRNA and these results were supported by Western blot analysis of protein levels following IL-10 treatment. These data suggest that IL-10 reduces inflammation and that targeting injury-induced inflammation is an effective strategy for limiting the extent of neuronal damage following excitotoxic SCI and thus the onset and progression of injury-induced pain behaviors.

Lumbar transplant of neurons genetically modified to secrete brain-derived neurotrophic factor attenuates allodynia and hyperalgesia after sciatic nerve constriction.

Chronic delivery of anti-nociceptive molecules by means of cell grafts near the pain processing centers of the spinal cord is a newly developing technique for the treatment of neuropathic pain. The rat neuronal cell line, RN33B, derived from E13 rat brainstem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat brain-derived neurotrophic factor cDNA (BDNF), and the BDNF-synthesizing cell line, 33BDNF.4, was isolated. The 33BDNF.4 cells synthesized mature BDNF protein at permissive temperature (33 degrees C), when the cells were proliferating, and during differentiation at non-permissive temperature (39 degrees C) in vitro. The bio-active BDNF protein was also secreted by the cells during both growth conditions, as measured by ELISA analysis of BDNF content and secretion. The bio-activity of the BDNF in 33BDNF.4 cell conditioned media was assessed by neurite outgrowth from E15 dorsal root ganglion (DRG) cultures. A control cell line, 33V1, transfected with the vector alone, did not synthesize or secrete any significant BDNF at either growth condition. Both cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hindpaw. When 33BDNF.4 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord and the 33BDNF.4 cells continued to synthesize BDNF in vivo. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2-7 week period after grafts of 33BDNF.4 cells. The maximal effect on chronic pain behaviors with the BDNF grafts occurred 2-3 weeks after transplant and the anti-nociceptive effects of the BDNF cell grafts was permanent. Transplants of the control 33V1 cells had no effect on the allodynia and hyperalgesia induced by CCI and these cells did not synthesize BDNF in vivo. These data suggest that a chronically applied, low local dose of BDNF supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver anti-nociceptive molecules, such as BDNF, in a model of chronic pain offers a novel approach to pain management and such 'biologic minipumps' can be developed for safe use in humans.

Transplants of neuronal cells bioengineered to synthesize GABA alleviate chronic neuropathic pain.

The use of cell lines utilized as biologic "minipumps" to provide antinociceptive molecules, such as GABA, in animal models of pain is a newly developing area in transplantation biology. The neuronal cell line, RN33B, derived from E13 brain stem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat GAD67 cDNA (glutamate decarboxylase, the synthetic enzyme for GABA), and the GABAergic cell line, 33G10.17, was isolated. The 33G10.17 cells transfected with the GAD67 gene expressed GAD67 protein and synthesized low levels of GABA at permissive temperature (33 degrees C), when the cells were proliferating, and increased GAD67 and GABA during differentiation at nonpermissive temperature (39 degrees C) in vitro, because GAD67 protein expression was upregulated with differentiation. A control cell line, 33V1, transfected with the vector alone, contained no GAD67 or GABA at either temperature. These cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and tactile hyperalgesia, were evaluated after CCI in the affected hind paw. When 33G10.17 and 33V1 cells were transplanted in the lumbar subarachnoid space of the spinal cord 1 week after CCI, they survived greater than 7 weeks on the pia mater around the spinal cord. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced during the 2-7-week period after grafts of 33G10.17 cells. The maximal effect on chronic pain behaviors with the GABAergic grafts occurred 2-3 weeks after transplantation. Transplants of 33V1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of GABA presumably supplied by transplanted cells near the spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of neural cell lines that are able to deliver inhibitory neurotransmitters, such as GABA, in a model of chronic pain offers a novel approach to pain management.

Lumbar transplant of neurons genetically modified to secrete galanin reverse pain-like behaviors after partial sciatic nerve injury.

The use of cell lines as biologic "minipumps" to chronically deliver antinociceptive molecules such as the peptide galanin near the pain processing centers of the spinal cord after nerve injury is a newly developing technology for the treatment of neuropathic pain. The neuronal rat cell line, RN33B, derived from E13 brainstem raphe and immortalized with the SV40 temperature-sensitive allele of large T antigen (tsTag), was transfected with rat preprogalanin (GAL) cDNA and the galanin-synthesizing and -secreting cell line, 33GAL.19, was isolated [1]. The 33GAL.19 cells transfected with the GAL gene expressed immunoreactivity (ir) for the GAL protein and synthesized low levels of GAL-ir at permissive temperature (33 degrees C), when the cells were proliferating, and increased GAL-ir during terminal differentiation at non-permissive temperature (39 degrees C) in vitro. A control cell line, 33V.1, RN33B cells transfected with the pCEP4 vector alone and similarly isolated by subcloning, contained no detectible GAL-ir at either temperature in vitro. These cell lines were used as grafts in a model of chronic neuropathic pain induced by unilateral chronic constriction injury (CCI) of the sciatic nerve. Pain-related behaviors, including cold and tactile allodynia and thermal and mechanical hyperalgesia, were evaluated in the affected hindpaw after CCI and transplants. The 33GAL.19 and 33V.1 cells transplanted in the lumbar subarachnoid space near the spinal cord one week after CCI, survived at least seven weeks on the pial surface around the spinal cord and only the 33GAL.19 cells expressed GAL-ir in vivo after transplant. Furthermore, the tactile and cold allodynia and tactile and thermal hyperalgesia induced by CCI was significantly reduced or eliminated during the two to seven week period after grafts of 33GAL.19 cells. The maximal effect on chronic pain behaviors with the GAL grafts occurred one to three weeks after transplantation. Transplants of 33V.1 control cells had no effect on the allodynia and hyperalgesia induced by CCI. These data suggest that a chronically applied, low local dose of galanin supplied by transplanted cells near the lumbar spinal dorsal horn was able to reverse the development of chronic neuropathic pain following CCI. The use of transplants of genetically modified neural cell lines that are able to deliver antinociceptive molecules, such as galanin, offers a safe and novel approach to pain management.

Differential regulation of synaptic vesicle protein genes by target and synaptic activity.

Differentiation of presynaptic nerve terminals involves changes in gene expression; these may be regulated by synaptic transmission and/or by contact with the target muscle. To gain insight into the control of presynaptic differentiation, we examined the regulation by target and synaptic activity of synaptic vesicle protein (SVP) genes in the chick ciliary ganglion (CG). In the CG, two SVP genes, synaptotagmin I (syt I) and synaptophysin II (syp II), are coordinately upregulated at the time of target contact. To test the hypothesis that this upregulation is induced by target contact, we examined mRNA levels of syt I and syp II in CGs from embryos in which one eye had been removed before axon outgrowth. As expected, target removal prevented the normal upregulation of syt I mRNA in the deprived ganglion. In contrast, and unexpectedly, syp II mRNA upregulation was not affected. The target dependence of syt I upregulation was not attributable to nerve-muscle transmission, because blockade of this transmission had no effect on SVP mRNA levels. Surprisingly, blockade of synapses onto CG neurons from the brain also did not affect syt I mRNA levels but increased levels of syp II mRNA. We conclude that contact with target induces upregulation of syt I mRNA, which is the case for spinal motor neurons. However, the normal upregulation of syp II mRNA is not controlled by the same signal(s). Instead, our results suggest that these two SVP genes are differentially regulated, both by target contact and by blockade of synaptic transmission.

Changes in GAD- and GABA- immunoreactivity in the spinal dorsal horn after peripheral nerve injury and promotion of recovery by lumbar transplant of immortalized serotonergic precursors.

We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize 5HT and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for 5HT and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter gamma-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1-8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered 5HT levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.

Dynamic interactions between nerve and muscle in Caenorhabditis elegans.

Synaptogenesis among developing motoneurons and muscles was examined in the nematode Caenorhabditis elegans. In this animal embryonic precursor cells give rise to regionally localized, contiguous clones of muscle cells that form two dorsal and two ventral sets that run longitudinally along the body wall. Ablation of selected embryonic muscle precursors resulted in gaps in the posterior dorsal muscle quadrants. We compared the morphological development of GABAergic locomotory neurons in the presence and absence of their target muscle cells. The results led to four main conclusions: (1) target muscle cells are not required for the morphological differentiation of the motoneurons; (2) target muscle cells appear to be required for the formation of presynaptic varicosities by the motoneurons; (3) embryonic muscle cells serve as a guide for migrating postembryonic muscle cells and in the absence of these guides the postembryonic muscles often assume ectopic locations; and (4) in the presence of ectopic muscle cells, the GABAergic locomotory neurons sprouted and formed branches that contributed to ectopic neuromuscular junctions.

Genetic transformation of the synaptic pattern of a motoneuron class in Caenorhabditis elegans.

Caenorhabditis elegans possesses two classes of inhibitory locomotory neurons, the DD and VD motoneurons (mns), and they form complementary components of a cross-inhibitory neuronal network innervating dorsal and ventral body muscles. The DD and VD mns (collectively called the D mns) share a number of morphological and neurochemical features, and mutations in a number of different genes disrupt both cell types in identical ways; however, the DD and VD mns have different lineal origins and different synaptic patterns. Given the number of phenotypic features shared by the D mns, it was of interest to determine what is responsible for the synaptic patterns that distinguish them. An analysis of the locomotory defect along with a genetic epistasis test suggested that unc-55 mutations alter the function of the VD but not the DD mns. Correlated with the defective locomotory behavior of unc-55 mutants was an alteration in the distribution of varicosities, structures associated with presynaptic elements, on the VD mns. The pattern of varicosities of the unc-55 VD mns resembled that of the wild-type DD mns. Moreover, the selective removal of the DD mns revealed that unc-55 VD mns had adopted a functional role appropriate for the DD mns. Thus, unc-55 appears to be involved in producing the synaptic patterns that distinguish the two D mn classes from one another; when the gene is mutated the VD and DD mns become structurally similar and functionally equivalent.

Changing synaptic specificities in the nervous system of Caenorhabditis elegans: differentiation of the DD motoneurons.

During postembryonic development, the DD motoneurons in the nematode Caenorhabditis elegans completely reorganize their pattern of synapses. Ablation of a pair of embryonic precursors results in the absence of this entire class of motoneurons. In their absence animals exhibit two developmentally distinct locomotory defects. The transition period from one defect to the other is correlated with the synaptic reorganization of the DD mns. Mutations in a gene (unc-123) have been isolated that exhibit locomotory defects similar to those of the ablated adult animals. Genetic and cellular analyses of one of these alleles suggest that the unc-123 gene product may be involved in the reestablishment of functional synapses in these neurons.

A role for carbonic anhydrase in early eye morphogenesis.

The carbonic anhydrase inhibitor methazolamide was used to test the hypothesis that carbonic anhydrase activity plays a role in the early morphogenesis of the chick eye. Methazolamide was administered topically to eyes of 4-7-day-old chick embryos in shell-free culture. Either continuous application of drug solution with a miniosmotic pump or a single daily application of drug resulted in significant reduction in embryonic eye growth relative to sham-treated controls.