Motoo Shinoda

Changes in Distribution of Cystatin C, Apolipoprotein E and Ferritin in Rat Hypothalamus after Hypophysectomy

To clarify the mechanism underlying the process of degeneration of injured CNS neurons, we have immunohistochemically examined the distribution of cystatin C, apolipoprotein E, IgG, transferrin and ferritin in the hypophysectomized rat hypothalamus. Stainings for ferritin revealed that reactive microglial cells massed in the paraventricular and supraoptic nuclei 14 days after hypophysectomy, when the degeneration of vasopressin neuronal cell bodies was apparent. Cystatin C‐positive magnocellular neurons first appeared at 4 days and the number of intensely‐stained cells increased rapidly up to the 7th day of hypophysectomy, followed by a decrease thereafter. Most of such cystatin C‐positive neurons were simultaneously stained with anti‐vasopressin serum. Accumulation of apolipoprotein E in extra‐cellular spaces was obvious in the both hypothalamic magnocellular nuclei at 7 days. Several apolipoprotein E‐positive cells were localized in the supraoptic nucleus, although the number of apolipoprotein E‐positive cells was much smaller than that of cystatin C‐positive cells. The experiments performed with the transferrin and IgG antibodies showed undetectable levels of such molecules in and around the degenerating magnocellular neurons during whole experimental periods. These findings suggest the importance of cystatin C and apolipoprotein E in the process of degeneration and/or regeneration of magnocellular neurons after hypophysectomy.

Meninges play a neurotrophic role in the regeneration of vasopressin nerves after hypophysectomy

Following hypophysectomy the regenerating fibers of magnocellular neurons are known to establish new neurohemal connections with reorganized vasculatures in the median eminence, which lead to establishment of a posterior pituitary-like structure. In order to examine the role of the meninges (the pia mater and the arachnoid) in this regeneration process, we implanted the meningeal tissues obtained from neonatal rat pups into the third ventricle of the adult rats, and then hypophysectomized the host animals. Ten days after hypophysectomy, the meningeal tissue grafts were found to be densely innervated by regenerating vasopressin-immunoreactive fibers. Such fibers had dots and frequently formed large punctuations. On the contrary, few vasopressin fibers were found within the cortical tissue grafts. Further, the exposure of primary hypothalamic cell cultures to the medium conditioned by meningeal cell cultures promoted not only the survival of vasopressin-immunoreactive neurons but also the outgrowth and aborization of the neurites. The survivals of cortical and cerebellum neurons in culture were also promoted by the conditioned medium. These findings raise the possibility that the meninges play an important role in the axonal regeneration process after hypophysectomy.

Effect of 2-deoxy-d-glucose on acetylcholine and histamine levels in gastric juice of pylorus-ligated rats anesthetized with urethane

Acetylcholine (ACh) in gastric juice was detected and measured by pretreatment of acetylcholinesterase inhibitor, 1 mM eserine (1 ml/rat, p.o.), in pylorus-ligated rats, by liquid chromatography with electrochemical detection. In order to elucidate whether or not the ACh level in gastric juice reflects the activity of cholinergic neurons, the effect of 2-deoxy-D-glucose (2-DG), a vagus stimulant, on the levels of ACh, histamine and gastric acid in gastric juice was investigated in pylorus-ligated rats anesthetized with urethane (1.25 g/kg, i.p.). Under the non-anesthetic condition, ACh, histamine and gastric acid levels were 100+/-25 pmol/h, 120+/-10 ng/h, and 240+/-32 microequiv./h, respectively. These levels were completely inhibited by urethane anesthesia. Under the anesthetized condition, 2-DG (50-200 mg/kg, i.v.) significantly increased ACh and histamine levels in gastric juice, as well as acid secretion. The 2-DG (200 mg/kg, i.v.)-induced increases in these levels were completely inhibited by vagotomy. These results suggest that ACh level measured in gastric juice reflects the activity of cholinergic transmission. Furthermore, these results also support the conclusion that vagus stimulation facilitates not only cholinergic transmission but also histaminergic transmission related to gastric acid secretion.

APPEARANCE OF BETA-2-MICROGLOBULIN IN RAT HYPOTHALAMIC MAGNOCELLULAR NEURONS AFTER HYPOPHYSECTOMY

To clarify the relationship between neuronal cell degeneration and MHC class-I complex expression, we have immunohistochemically examined the distribution of beta(2)-microglobulin in the hypophysectomized rat hypothalamus. In the sham-operated control rats, positive stainings were distributed only in blood vessels in the hypothalamic areas where magnocellular neurons were localized. Three days after hypophysectomy, positive stainings appeared in a large number of magnocellular neurons in the paraventricular and supraoptic nuclei. Most of such beta(2)-microglobulin-positive cells were simultaneously stained with antivasopressin serum. The pattern of distribution of positive cells and the intensity of the stainings remained unchanged at least until the 14th day. These morphological findings suggest that the process of degeneration of hypothalamic magnocellular neurons after hypophysectomy is a useful model to investigate the role of MHC class I complex in the brain.