Effects of neutralizing antibodies to TNF-alpha on pain-related behavior and nerve regeneration in mice with chronic constriction injury

Inhibition of proinflammatory cytokines reduces hyperalgesia in animal models of painful neuropathy. We set out to investigate the consequences of this treatment for nerve regeneration. Here we examined the sequels of epineurial application of neutralizing antibodies to tumor necrosis factor-alpha (TNF) in chronic constriction injury (CCI) of the sciatic nerve in C57/BL 6 mice. The mice were tested behaviorally for manifestations of thermal hyperalgesia and mechanical allodynia. Nerve regeneration was assessed by morphometry of myelinated nerve fibers in the sciatic nerve and of the epidermal innervation density in the glabrous skin of the hindpaws. Antibodies to TNF reduced thermal hyperalgesia and mechanical allodynia after CCI. Myelinated fiber density in the sciatic nerve was reduced to 30% of normal on day 7 after surgery, and reached 60% on day 45, with no difference between antibody-treated and untreated animals. Epidermal innervation density as shown by PGP 9.5 and CGRP immunohistochemistry was reduced to 25-47% at both time points after CCI, again without differences between antibody treated and untreated mice. Myelinated fiber density but not epidermal innervation density was correlated to thermal and mechanical withdrawal thresholds. We conclude that neutralization of endoneurial TNF attenuates pain related behavior but has no effect on nerve regeneration. Furthermore, the number of epidermal nerve fibers is not relevant to the magnitude of behavioral hyperalgesia in CCI.     

Expression and regulation of tyrosine hydroxylase in adult sensory neurons in culture: Effects of elevated potassium and nerve growth factor

To determine whether similar molecular mechanisms regulate the same proteins in diverse neuronal populations, the present study compared regulation of tyrosine hydroxylase (TOH) in placodal sensory and neural crest-derived sympathetic neurons in tissue culture. Long-term explant cultures of adult nodose and petrosal sensory ganglia (NPG) contained abundant TOH-immunoreactive neurons and exhibited TOH catalytic activity, as in vivo. After an initial decline during the first week of culture, enzyme activity was maintained at a stable plateau of 60% of zero time values for at least 3 weeks. However, exposure of 2-week-old cultures to depolarizing concentrations of potassium (K+; 40 mM) increased TOH activity approximately two-fold; total protein was unchanged, suggesting that the rise was due to increased TOH specific activity. Therefore, membrane depolarization in vitro appears to regulate this specific catecholaminergic (CA) trait in sensory, as in sympathetic CA cells. In sympathetic neurons, NGF regulates TOH activity throughout life. In marked contrast, TOH activity in adult NPG cultures was unchanged in the presence of 0, 10 or 100 units NGF/ml or in the presence of high concentrations of antiserum against the beta-subunit of NGF. Adult sympathetic neurons, however, grown under identical conditions, exhibited a 5- to 10-fold rise in TOH activity in the presence of NGF. Thus, unlike sympathetics, CA metabolism in adult NPG neurons is not regulated by NGF in vitro; NGF is therefore unlikely to mediate target effects on CA metabolism in placodal sensory neurons in vivo. Our findings indicate that certain mechanisms of CA regulation are shared by placodal sensory and neural crest-derived sympathetic neurons, whereas others are not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related decrease of nerve growth factor-like immunoreactivity in the basal forebrain of senescence-accelerated mice

The senescence-acceleratedmouse P10 (SAMP10) is a murine model of accelerated senescence characterized by the deterioration of learning and memory with advancing age. In the present study, we examined the distribution of nerve growth factor (NGF) immunohistochemically in SAMP10 mice and its control strain, SAMR1. In both strains, NGF-like immunoreactivity (NGF-IR) was observed in neurons throughout the entire forebrain and in glial cells in a particular location. In aged SAMP10 mice, each layer of the cerebral cortex retained its NGF-IR, although the thickness of the cortical mantle was markedly decreased in comparison with younger animals. There was an age-related decline in NGF-IR in the substantia innominata of SAMP10 mice at the age of 10 months, when compared to 2-month-old SAMP10. These results indicate age-related decrease of NGF in the basal forebrain in SAMP10 mice.     

Expression of human tyrosine hydroxylase-chloramphenicol acetyltransferase (CAT) fusion gene in the brains of transgenic mice as examined by CAT immunocytochemistry

Summary We have produced transgenic (Tg) mice carrying 5.0-kb fragment from the 5-flanking region of the human tyrosine hydroxylase (hTH) gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT) [Sasaoka et al. (1992) Mol Brain Res 16: 274–286]. In the brain of the Tg mice, CAT expression has been observed in catecholaminergic (CAnergic) neurons and also in non-CAnergic neurons. The aim of the present study is to examine in detail the cell-type specific expression of the hTH-CAT fusion gene in the brain of the Tg mice, by use of immunohistochemistry for CAT, TH, and aromatic L-amino acid decarboxylase (AADC). CAT-immunoreactive cells were found in CAnergic brain regions which contained TH-positive cells, and also in non-CAnergic brain regions which contained no TH-labeled cells. The non-CAnergic brain regions that represented CAT-stained cells were further divided into two groups: (i) regions containing AADC-labeled cells, for example, bed nucleus of the stria terminalis, nucleus suprachiasmaticus, mammillary body, nucleus raphe dorsalis, inferior colliculus, and nucleus parabrachialis, and (ii) regions containing no AADC-positive cells, for example, main olfactory bulb (except A16), accessory olfactory bulb, nucleus olfactorius anterior, caudoputamen, septum, nucleus accumbens, hippocampus, medial nucleus of the amygdala, entorhinal cortex, nucleus supraopticus, and parasubiculum. The results indicate that the 5.0-kb DNA fragment flanking the 5 end of the hTH gene may contain the element(s) specific for neuron-specific TH expression but which may be insufficient to attenuate ectopic expression.     

An acceleration of age-related increases in levels of the β-subunit of nerve growth factor in selected tissues from senescence-accelerated mice (SAM-P/8)

An investigation was made of age-related changes in levels of the β-subunit of nerve growth factor (β-NGF) in selected tissues and of testosterone in serum in senescence-accelerated mice (SAM-P/8) and in the control mice (senesence-resistant mice; SAM-R/1). The concentrations of testosterone in serum were higher in SAM-P/8 than in SAM-R/1 at ages 2 and 4 mo. The level of β-NGF in the thymus from SAM-R/1 increased with age, resulting in a statistically significant difference in its level between mice at ages 2 and 12 mo. By contrast, there was a transient increase in SAM-P/8 at around age 4 mo with a subsequent decrease. Consequently, significant differences were apparent in levels of β-NGF between the two types of mouse at ages 2 and 4 mo. Similar results were obtained in the adrenal gland and testis. Compared to SAM-R/1 at age 2 mo, the average concentrations of β-NGF in the hypophysis were higher in SAM-R/1 at ages 4 and 8 mo and in SAM-P/8 at all ages. In other tissues tested, no remarkable differences were detected. Our present results indicate that, in SAM-P/8, the elevation in levels of β-NGF in the thymus, adrenal gland, testis, and hypophysis occurs in the early period of life compared to the control mice. Possible dysfunction of the disorder of hypophysis is discussed.