Immunocytochemical demonstration of nerve growth factor and histofluorescence of catecholaminergic nerves in the salivary glands of diabetic mice

Summary Nerve growth factor (NGF) was localized in the submandibular, sublingual, and parotid salivary glands of male and female diabetic mice and their normal littermates by immunoperoxidase staining usingp-phenylenediamine-pyrocatechol as a chromogen for the cytochemical demonstration of peroxidase activity. In the normal male submandibular gland, immunoreactive NGF was localized in the apical regions of granular, intercalated and collecting duct cells, while in the normal female submandibular gland, NGF was present throughout the cytoplasm of granular duct cells. The localization of NGF in the diabetic male and female submandibular glands was similar and resembled that of the normal female. NGF immunoreactivity was also observed in the striated duct cells in the sublingual and parotid glands of all four types of mice.The sympathetic innervation of the submandibular glands of normal and diabetic mice was demonstrated using glyoxylic acid-induced histofluorescence. The pattern of sympathetic innervation and the intensity of catecholamine fluorescence was consistently different in the four types of mice. In the normal male submandibular gland the fluorescence was very intense, particularly in nerves adjacent to the granular ducts. In the normal female submandibular gland, the fluorescence was weak, while in the diabetic male and female the fluorescence was moderate.The correlation between the intensity of the immunocytochemical staining for NGF and the catecholamine fluorescence adjacent to the granular ducts suggests a trophic influence of the NGF-containing granular ducts on their sympathetic innervation.     

Reduction of nerve growth factor level in the brain of genetically ataxic mice (weaver, reeler)

By a highly sensitive enzyme immunoassay we measured the level of nerve growth factor (NGF) in the cerebellum and cerebrum of the neurologically mutant mice, weaver, reeler and Purkinje cell degeneration (PCD). A significant decrease in NGF level was observed in both cerebellum and cerebrum of weaver and reeler mutants of either sex. However, there was no such difference between normals and mutants in the case of the PCD mice. These results show that weaver and reeler mice have abnormalities of NGF synthesis and/or degradation not only in the cerebellum but also in the cerebrum.     

Decrease in the levels of NGF and BDNF in brains of mice fed a tryptophan-deficient diet

The roles of dietary tryptophan (Trp) were evaluated in regulation of production of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT)-3 in the various brain regions in ddY mice. Feeding the mice a Trp-deficient diet for 2 weeks significantly decreased in the hippocampal level of NGF but not those of BDNF and NT-3, as compared with feeding an adequate Trp diet. The mice fed excess Trp did not have different levels of any of these neurotrophins than in the mice fed an adequate Trp diet. The levels of BDNF in the cerebral cortex were also significantly lower in the mice fed on a Trp-deficient diet, while the levels of NGF and NT-3 in the region were not modulated upon feeding of the diet. The dietary Trp level had no significant effect on the levels of NGF, BDNF, or NT-3 in the entorhinal cortex nor septum of the mice. These results demonstrate that the brain levels of NGF and BDNF are dependent on the dietary content of tryptophan.     

Macrophage dependence of peripheral sensory nerve regeneration: possible involvement of nerve growth factor.

The levels of NGF and NGF receptor mRNA, the degree of macrophage recruitment, and the ability of sensory and motor axons to regenerate were measured in C57BL/Ola mice, in which Wallerian degeneration following a nerve lesion is very slow. Results were compared with those from C57BL/6J and BALB/c mice, in which degeneration is normal. We found that in C57BL/Ola mice, apart from the actual lesion site, recruitment of macrophages was much lower, levels of mRNA for both NGF and its receptor were raised only slightly above normal, and sensory axon regeneration was much impaired. Motor axons regenerated quite well. These results provide in vivo evidence that macrophage recruitment is an important component of NGF synthesis and of sensory (but not motor) axon maintenance and regrowth.     

Effect of Nerve Growth Factor in Ethylcholine Mustard Aziridinium (AF64A)-Treated Rats: Sensitization of Cholinergic Enzyme Activity in the Septohippocampal Pathway

Abstract: In this study, we examined the effects of nerve growth factor (NGF) administration on cholinergic enzyme activity in both normal and ethylcholine mustard aziridinium (AF64A)-treated rats. Choline acetyltransferase (ChAT) and acetylcholinesterase activity were measured in the hippocampus and septum of rats chronically administered NGF (0.36–2.85 µg/day) into the lateral ventricle for 14 days. In both normal and AF64A-treated rats, NGF increased cholinergic enzyme activity in a dose-dependent manner. Furthermore, although NGF increased ChAT activity in normal rats by 147%, it had a greater effect in AF64A-treated rats, increasing ChAT activity as much as 273%. NGF increased acetylcholinesterase activity in normal rats by only 125% but produced a 221% increase in this activity in AF64A-treated rats. These data indicate that AF64A produces an increased sensitivity to NGF in cholinergic neurons.     

Nerve growth factor (NGF) exerts its pro-apoptotic effect via the P75NTR receptor in a cell cycle-dependent manner.

Nerve growth factor (NGF), the prototypic member of the neurotrophin family of growth factors, exerts its action via two receptors, P75NTR and TrkA, the expression of which varies at the cell surface of neuroblastoma cells (SH-SY5Y cells) in a cycle phase-specific manner. NGF was pro-apoptotic on growing cells expressing preferentially P75NTR and exhibited a potent anti-apoptotic effect on quiescent cells, when TrkA was prevalent at the cell surface, showing that NGF can have a dual action on SH-SY5Y cells depending on the relative cell surface expression of TrkA and P75NTR. The pro-apoptotic activity of NGF but not its anti-apoptotic activity was abrogated by an antibody against the extracellular domain of P75NTR and in cell isolated from P75NTR knock-out mice indicating that NGF exhibits a proapoptotic activity via P75NTR exclusively. On the other hand, we showed that the anti-apoptotic activity of NGF was specifically mediated by an interaction with TrkA with no contribution of P75NTR, as demonstrated on SK-N-BE cells transfected with TrkA in which NGF was a potent anti-apoptotic compound but did not exhibit any pro-apoptotic activity. These results support the hypothesis that the survival response to NGF depends on its binding to TrkA without any involvement of P75NTR which in turn selectively mediates the pro-apoptotic activity of NGF with no contribution of TrkA and show that, depending on the growth state of the cells, NGF exhibits dual pro- or anti-apoptotic properties via P75NTR and TrkA, respectively.     

CHANGES OF ENZYME PATTERN IN THE SYMPATHETIC NERVOUS SYSTEM OF ADULT MICE AFTER SUBMAXILLARY GLAND REMOVAL; RESPONSE TO EXOGENOUS NERVE GROWTH FACTOR

—Removal of the submaxillary glands, the apparent site of NGF synthesis in adult mice, caused a decrease in the activity of all the enzymes involved in the biosynthesis of noradrenaline in the peripheral sympathetic nervous system. Thus, tyrosine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) DOPA decarboxylase (EC 4.1.1.28.) and dopamine β-hydroxylase (EC 1.14.17.1.) showed reduced activity 10 days after removal of the submaxillary glands in both superior cervical and stellate ganglia. This decrease in enzyme activity persisted up to 100 days after surgery. The fourth enzyme studied, choline acetyl-transferase (EC 2.3.1.6.) which is exclusively localized within the presynaptic cholinergic terminals of the ganglia was not affected by sialectomy. A dose of 50 μg NGF/animal/day given over 4 days was only able to restore the enzyme activity to control levels in the superior cervical ganglia of sialectomized mice whereas in stellate ganglia the enzyme activities rose above control levels to a similar extent in sialectomized and non-sialectomized animals. These results provide biochemical evidence that NGF may play a role not only during the growth and normal development of the peripheral sympathetic nervous system but also in the maintenance of its functional integrity in the adult animal.     

Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain

Abstract: Effects of thyroxine (T₄) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T₄ caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T₄ was effective on the post-natal rats only up to day 11. These results suggest that T₄ plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T₄ suggest that the effects of T₄ on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T₄.     

Ablation of TrkA function in the immune system causes B cell abnormalities

The nerve growth factor (NGF) receptor TrkA is widely expressed in non-neural tissues suggesting pleiotropic functions outside the nervous system. Based on pharmacological and immuno-depletion experiments, it has been hypothesized that NGF plays an important role in the normal development and function of the immune system. However, attempts to unravel these functions by conventional gene targeting in mice have been hampered by the early postnatal lethality caused by null mutations. We have developed a novel 'reverse conditional' gene targeting strategy by which TrkA function is restored specifically in the nervous system. Mice lacking TrkA in non-neuronal tissues are viable and appear grossly normal. All major immune system cell populations are present in normal numbers and distributions. However, mutant mice have elevated serum levels of certain immunoglobulin classes and accumulate B1 cells with aging. These data, confirmed in a classical reconstitution model using embryonic fetal liver from TrkA-null mice, demonstrate that endogenous NGF modulates B cell development through TrkA in vivo. Furthermore, they demonstrate that many of the dramatic effects previously reported by pharmacological or immuno-depletion approaches do not reflect physiological developmental roles of TrkA in the immune system.     

Populations of NGF-dependent neurones differ in their requirement for BAX to undergo apoptosis in the absence of NGF/TrkA signalling in vivo.

Reports that apoptosis within populations of neurotrophin-dependent neurones is virtually eliminated in BAX-deficient mice and that BAX-deficient neurones survive indefinitely in culture without neurotrophins have led to the view that BAX is required for the death of neurotrophin-deprived neurones. To further examine this assertion in vivo, we have studied two populations of NGF-dependent neurones during the period of naturally occurring neuronal death in mice that lack BAX, NGF or the NGF receptor TrkA, alone and in combination. In the superior cervical ganglion (SCG), naturally occurring neuronal death and the massive loss of neurones that took place in the absence of NGF or TrkA were completely prevented by elimination of BAX. However, in the trigeminal ganglion, naturally occurring neuronal death was only partly abrogated by the elimination of BAX, and although the massive neuronal death that took place in this ganglion in the absence of NGF or TrkA was initially delayed in embryos lacking BAX, this subsequently occurred unabated. Accordingly, BAX-deficient neurones survived in defined without NGF whereas BAX-deficient trigeminal neurones died in the absence of NGF. These results indicate that whereas BAX is required for the death of SCG neurones during normal development and when these neurones are deprived of NGF/TrkA signalling in vivo, the death of trigeminal ganglion neurones occurs independently of BAX when they are deprived of NGF/TrkA signalling. We conclude that BAX is not universally required for neuronal death induced by neurotrophin deprivation, but that there are major differences for the requirement for BAX among different populations of NGF-dependent neurones.     

Role for nerve growth factor in the in vivo regulation of glutathione in response to LPS in mice

Since the redox state regulator glutathione (GSH), which influences lipopolysaccharide (LPS) anorexia, may be controlled by cytokines, we studied the roles of tumour necrosis factor-alpha (TNFalpha) and nerve growth factor (NGF) in the GSH response to intraperitoneal (ip) LPS injection in mice. Basal NGF and total reduced GSH (trGSH) levels were up-regulated in brain and liver of TNFalpha-knock-out (KO) mice, and this was associated with attenuated LPS anorexia. The increases in NGF and trGSH presumably contributed to the attenuated anorexia in response to LPS because transgenic mice over-expressing NGF (NGF-tg mice) also had increased trGSH levels and displayed attenuated anorexia compared to the corresponding wild type (WT) mice. Attenuated LPS anorexia in NGF-tg mice was accompanied by reduced serum TNFalpha and IFNgamma levels compared to WT mice. In response to a second injection of LPS, NGF and trGSH levels, but not TNFalpha levels changed. This suggests that in vivo tissue trGSH changes following LPS in LPS-na?ve or LPS-pretreated mice are regulated by NGF rather than TNFalpha. The finding that genetic TNFalpha deficiency did not inhibit the acute trGSH response to LPS supports this interpretation. In sum, the results indicate i) that a decrease or increase in NGF is accompanied by a decrease or increase in trGSH levels and ii) that elevated NGF and/or trGSH levels attenuate some of the responses to LPS such as anorexia and cytokine production.     

Ventricular injection of nerve growth factor increases dopamine content in the striata of MPTP-treated mice

Experimental depletion of dopaminergic striatal neurons was induced in mice with the neurotoxin MPTP. To investigate a possible effect of nerve growth factor on the damaged neurons, we injected 4 g into the right cerebral ventricle of mice three days after the last administration of MPTP. We found a significant increase of dopamine and homovanillic acid in the striatum of MPTP treated mice after NGF administration when compared with dopamine and HVA levels in MPTP-treated control mice (p<0.001). The increase of dopamine and homovanillic acid seems to be related to a partial restorative effect of NGF on the damaged dopaminergic cells, since ventricular administration of NGF to normal mice did not increase dopamine or homovanillic acid contents above the levels measured in untreated controls. It appears that administration of nerve growth factor prcduces a beneficial effect on damaged dopaminergic neurons; this effect could be due to stimulation of neuron sprouting from neurons that survived the toxic effect of MPTP. The increase of dopamine levels was seen 8 days after injection of nerve growth factor and was maintained at least until day 25, showing a lasting persistence of the restorative effect.     

江浙蝮蛇(Agkistrodon halys Pallas)蛇毒神经生长因子的分离纯化和特性

神经生长因子（ｎｅｒｖｅ ｇｒｏｗ ｔｈ ｆａｃｔｏｒ, Ｎ Ｇ Ｆ）是第一个被发现,也是迄今为止研究得最为清楚的一种神经营养因子 利用 Ｐ Ｃ１２ 细胞生物活力测定为跟踪检测手段,分别经过 Ｃ Ｍ Ｓｅｐｈａｒｏｓｅ Ｃ Ｌ ６ Ｂ、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 及 Ｆ Ｐ Ｌ Ｃ ｍ ｏｎｏ Ｓ层析,从３０ ｇ 江浙蝮蛇粗毒中分离纯化到２００ μｇ Ｎ Ｇ Ｆ,纯化倍数高达１０５经 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 测定,该蛋白分子量为 ２６ ｋ Ｄ,由两个亚基通过二硫键交联组成二体形式等电聚焦显示其等电点为６７,与氨基酸组成分析结果相吻合 江浙蝮蛇神经生长因子的生物活力水平与小鼠２５ Ｓ Ｎ Ｇ Ｆ相当,在１～１００ μｇ／ Ｌ 的浓度范围内维持 Ｐ Ｃ１２ 细胞在无血清条件下的存活     

Effect of quantity and quality of dietary protein on choline acetyltransferase and nerve growth factor, and their mRNAs in the cerebral cortex and hippocampus of rats

The brain protein synthesis is sensitive to the dietary protein; however, the role of dietary protein on biomarkers including choline acetyltransferase and nerve growth factor (NGF) for the function of cholinergic neurons remains unknown in young rats. The purpose of this study was to determine whether the quantity and quality of dietary protein affects the concentration of NGF and activity of choline acetyltransferase, and their mRNA levels in the brains of young rats. Experiments were carried out on five groups of young rats (4 weeks) given the diets containing 0, 5, 20% casein, 20% gluten or 20% gelatin for 10 days. The activity of choline acetyltransferase in the cerebral cortex and hippocampus declined gradually with a decrease in quantity and quality of dietary protein. The concentration of NGF in the cerebral cortex and the mRNA levels of choline acetyltransferase in the cerebral cortex and hippocampus did not differ among groups. However, the concentration and mRNA level of NGF in the hippocampus was significantly lower in rats fed with lower quantity of protein or lower quality of protein. In the hippocampus, the mRNA levels of NGF significantly correlated with the NGF concentration when the quantity (r = 0.704, P < 0.01) and quality (r = 0.682, P < 0.01) of dietary protein was manipulated. It was further found that a significant positive correlation existed between the NGF concentration and the activity of choline acetyltransferase in the hippocampus (dietary protein quantity, r = 0.632, P < 0.05; dietary protein quality, r = 0.623, P < 0.05). These results suggest that the ingestion of lower quantity and quality of dietary protein are likely to control the mRNA level and concentration of NGF, and cause a decline in the activity of choline acetyltransferase in the brains of young rats.     

神经生长因子对兔坐骨神经损伤后脊髓前角运动神经元乙酰胆碱酯酶的影响

钳夹损伤兔右坐骨神经,于损伤处注射蛇毒ＮＧＦ４００Ｂｕ／ｋｇ／日,损伤术后１,３,７天和２,３,４,６,８周动态观察脊髓腰段伤侧第Ⅸ板层外侧群的大型运动神经元的ＡＣｈＥ活性改变。结果表明术后１,３天实验组（指损伤给药组）和对照组（指损伤对照组）ＡＣｈＥ活性均下降（Ｐ＞００５）;术后１,２,３周对照组ＡＣｈＥ活性明显下降,而实验组ＡＣｈＥ活性逐渐趋于恢复（Ｐ＜００１）;术后６周实验组ＡＣｈＥ活性恢复至正常水平（Ｐ＜００１）。本研究显示蛇毒ＮＧＦ对坐骨神经损伤后脊髓前角运动神经元ＡＣｈＥ活性恢复有促进作用,从而对运动神经元可起一定的保护作用和促进恢复的作用     

p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength

During muscle development, the p75(NTR) is expressed transiently on myoblasts. The temporal expression pattern of the receptor raises the possibility that the receptor is influencing muscle development. To test this hypothesis, p75(NTR)-deficient mutant mice were tested for muscle strength by using a standard wire gripe strength test and were found to have significantly decreased strength relative to that of normal mice. When normal mybolasts were examined in vivo for expression of NGF receptors, p75(NTR) was detected on myoblasts but the high affinity NGF receptor, trk A, was not co-expressed with p75(NTR). In vitro, proliferating C2C12 and primary myoblasts co-expressed the p75(NTR) and MyoD, but immunofluorescent analysis of primary myoblasts and RT-PCR analysis of C2C12 mRNA revealed that myoblasts were devoid of trk A. In contrast to the cell death functions that characterize the p75(NTR) in neurons, p75(NTR)-positive primary and C2C12 myoblasts did not differentiate or undergo apoptosis in response to neurotrophins. Rather, myoblasts survived and even proliferated when grown at subconfluent densities in the presence of the neurotrophins. Furthermore, when myoblasts treated with NGF were lysed and immunoprecipitated with antibodies against phosphorylated I-kappaB and AKT, the cells contained increased levels of both phospho-proteins, both of which promote cell survival. By contrast, neurotrophin-treated myoblasts did not induce phosphorylation of Map Kinase p42/44 or p38, indicating the survival was not mediated by the trk A receptor. Taken together, the data indicate that the p75(NTR) mediates survival of myoblasts prior to differentiation and that the activity of this receptor during myogenesis is important for developing muscle.     

Sexual difference in kininase activity in the mouse submandibular gland

A marked sexual difference in kininase activity was found in the adult mouse submandibular gland. The activity was over 3-fold higher in females than in males between 10 and 12 weeks of age. Castration of male mice increased kininase activity up to the level of females. Testosterone administration to castrated males restored enzyme activity to about the normal level. Moreover, testosterone administration to normal females decreased enzyme activity to about the level of normal male mice, while ovariectomy had no effect. These results suggest that kininase activity in the mouse submandibular gland is suppressively regulated by endogenous androgen.     

Comparison of nerve growth factor mRNA expression in cardiac and skeletal muscle in streptozotocin-induced diabetic mice

To address the role of nerve growth factor (NGF) in diabetes mellitus (DM)-induced cardiac autonomic neuropathy, we quantitated and compared the expression of NGF mRNA in the cardiac and the skeletal muscle in experimental DM mice with the RT-PCR-HPLC method, which we have developed previously, using a NGF deletion mutant RNA as an internal standard. DM was induced in ICR mice via intraperitoneal injection of streptozotocin. RT-PCR was performed using total RNA extracted from left ventricle and soleus muscle, and the levels of NGF mRNA were quantitated by HPLC analysis. NGF mRNA content of the cardiac muscle was 17-fold higher than the skeletal muscles in control mice. NGF mRNA content of the cardiac muscle in diabetic mice at 6 weeks was 4.0-fold higher than that in the control mice, while that of the skeletal muscle in diabetic mice was not different from the controls. These results indicated that the DM-induced increase in NGF mRNA content was higher in cardiac muscle than skeletal muscle, and that NGF might play an important role in cardiac autonomic neuropathy.