Mast cells are necessary for the hypothermic response to LPS-induced sepsis.

As central nervous system residents, mast cells contain many cytokines and are localized primarily near large blood vessels in the diencephalon and within the leptomeninges, making them candidates for immune to neural "cross talk." Using mast cell-deficient Kit(W-sh/W-sh) mice, we assessed the role of these cells in the thermoregulatory component of the immune response to lipopolysaccharide (LPS). Kit(W-sh/W-sh) and wild-type (WT) mice differed in several respects in response to injection of a high dose of LPS (1 mg/kg ip). Core temperature (T(c)) of WT mice decreased by approximately 3 degrees C, whereas Kit(W-sh/W-sh) mice did not become hypothermic but instead exhibited pronounced low-frequency T(c) oscillations around their baseline temperature. In addition, Kit(W-sh/W-sh) mice had lower levels of whole brain TNF-alpha but no differences in IL-1beta, IL-6, IFN-gamma, or histamine compared with WT mice following injection of the high dose of LPS, consistent with the role of TNF-alpha in sepsis. Kit(W-sh/W-sh) mice had increased resistance to LPS, and some survived a dose of LPS that was lethal in littermate controls. In contrast, Kit(W-sh/W-sh) and WT mice were similar in other aspects, namely, in the hyperthermia following injection of TNF-alpha (1.5 microg icv), reduced nighttime T(c) and locomotor activity (to 1 mg/kg LPS), response to a low dose of LPS (10 microg/kg ip), and response to subcutaneous turpentine injection. These results indicate that mast cells play a role in the regulation of thermoregulatory responses and survival following sepsis induction and suggest a brain site of action.

Mast cell stabilization limits hypoxic-ischemic brain damage in the immature rat.

Perinatal hypoxic-ischemic (HI) brain damage is a major cause of mortality and neurological morbidity in infants and children. Using an established model of unilateral hypoxia-ischemia in neonatal rats, the present study focused on mast cells (MCs), important regulators of inflammatory processes, as potential contributors to HI damage. MCs are present in the pia of the neonatal rat, entering the central nervous system (CNS) during cerebral development along penetrating blood vessels. Following hypoxia-ischemia, MC numbers increased dramatically in the ipsilateral (ischemic) hemisphere (p < 0.01). In animals exposed to hypoxia only, the numbers of MCs were elevated in both hemispheres to an extent equal to that observed in the contralateral hemisphere of HI animals (p < 0.05 vs. control). Within damaged areas (ipsilateral only), MCs were observed in regions of activated microglia and astroglia that characterize the ischemic hemisphere. Using a triple-label paradigm, MCs were observed along elongating blood vessels, some of which express the GLUT1 isoform of the glucose transporter protein, indicative of blood-brain barrier vessels. To determine whether MC activation has a role in HI brain damage, rat pups were treated with the MCs stabilizer, disodium cromoglycate (cromolyn), prior to and/or following hypoxia-ischemia. The cromolyn treatment inhibited MC migration into the CNS (p < 0.05) and limited brain damage more than 50% (p < 0.01) vs. saline controls. These data support the hypothesis that MCs are key contributors to the extent of brain damage due to hypoxia-ischemia in the immature animal.

Brain mast cell relationship to neurovasculature during development.

Mast cells, derived from the hematopoietic stem cell, are present in the brain from birth. During development, mast cells occur in two locations, namely the pia and the brain parenchyma. The current hypothesis regarding their origin states that brain mast cells (or their precursors) enter the pia and access the thalamus by traveling along the abluminal wall of penetrating blood vessels. The population in the pia reaches a maximum at postnatal (PN) day 11, and declines rapidly thereafter. Chromatin fragmentation suggests that this cell loss is due to apoptosis. In contrast, the thalamic population expands from PN8 to reach adult levels at PN30. Stereological analysis demonstrates that mast cells home to blood vessels. More than 96% of mast cells are inside the blood-brain barrier, with ~90% contacting the blood vessel wall or its extracellular matrix. Mast cells express alpha4 integrins -- a potential mechanism for adhesion to the vascular wall. Despite the steady increase in the volume of microvasculature, at all ages studied, mast cells are preferentially located on large diameter vessels (>16 microm; possibly arteries), and contact only those maturing blood vessels that are ensheathed by astroglial processes. Mast cells not only home to large vessels but also maintain a preferential position at branch points, sites of vessel growth. This observation presents the possibility that mast cells participate in and/or regulate vasculature growth or differentiation. The biochemical and molecular signals that induce mast cell homing in the CNS is an area of active investigation.

Brain mast cells are influenced by chemosensory cues associated with estrus induction in female prairie voles (Microtus ochrogaster).

Historically, the brain has been viewed as protected from the infiltration of peripheral hematopoietic cells by the blood-brain barrier. However, numerous immune cell types have been found in the central nervous system (CNS). Mast cells, granulocytic immune cells, are found in the CNS of birds and mammals and their numbers and location are influenced by both extrinsic and intrinsic factors, including reproductive behavior and endocrine status. The present study used female prairie voles (Microtus ochrogaster) to investigate the interactions between brain mast cells and stimuli associated with estrus induction. Unlike spontaneous ovulators such as rats and mice, female prairie voles are induced into estrus by chemosensory stimuli present in conspecific male urine. Prior to estrus induction, female voles have undetectable concentrations of estrogen that rise rapidly following exposure to a male or male urine. In the first experiment, we examined whether mast cells may be influenced by estrus induction. Female voles exposed to conspecific male urine had increased numbers of mast cells in the main olfactory bulbs and epithalamus (medial habenula), but not the thalamus or median eminence, relative to control groups. Next, to determine if this mast cell increase was the result of elevated estrogen concentrations, female voles were injected with estradiol or vehicle and brain mast cell numbers analyzed. No differences in brain mast cell numbers were observed between estradiol-injected and control females in any brain area investigated. Together, these results lend further support to the contention that mast cell numbers and/or distribution can be influenced by reproductively relevant stimuli and underscore the utility of this vole model for delineating the function of brain mast cells.

Mast cells in the rat brain synthesize gonadotropin-releasing hormone.

Mast cells occur in the brain and their number changes with reproductive status. While it has been suggested that brain mast cells contain the mammalian hypothalamic form of gonadotropin-releasing hormone (GnRH-I), it is not known whether mast cells synthesize GnRH-I de novo. In the present study, mast cells in the rat thalamus were immunoreactive to antisera generated against GnRH-I and the GnRH-I associated peptide (GAP); mast cell identity was confirmed by the presence of heparin, a molecule specific to mast cells, or serotonin. To test whether mast cells synthesize GnRH-I mRNA, in situ hybridization was performed using a GnRH-I cRNA probe, and the signal was identified as being within mast cells by the binding of avidin to heparin. GnRH-I mRNA was also found, using RT-PCR, in mast cells isolated from the peritoneal cavity. Given the function of GnRH-I in the regulation of reproduction, changes in the population of brain GnRH-I mast cells were investigated. While housing males with sexually receptive females for 2 h or 5 days resulted in a significant increase in the number of brain mast cells, the proportion of mast cells positive for GnRH-I was similar to that in males housed with a familiar male. These findings represent the first report showing that mast cells synthesize GnRH-I and that the mast cell increase seen in a reproductive context is the result of a parallel increase in GnRH-I positive and non-GnRH-I positive mast cells.

Stimuli from conspecifics influence brain mast cell population in male rats.

It is well established that mast cells occur within the brain of many species, and that the brain mast cell population is not static, but changes with the behavioral and physiological state of the animal. In this study, we tested whether exposure to conspecifics alters the number of brain mast cells in male rats, and then investigated the nature of stimuli influencing the changes observed in the number and localization of brain mast cells. Five days of cohabitation with an ovariectomized, estrogen-progesterone (OVX + EP)-treated female resulted in the largest number of thalamic mast cells, while pairing with such a female physically separated by a wire mesh or with a novel male produced a smaller, but significant increase over other pairings (OVX females for 5 days, OVX and OVX + EP females for 1 day, familiar or isolated males for 5 days). In all groups, mast cells were localized within specific dorsal thalamic nuclei, including the paraventricular nucleus, anterior nuclear group, or mediodorsal, ventroposterior, or medial geniculate nuclei. The results suggest that the behavioral and/or endocrine factors associated with cohabitation with conspecifics are sufficient to alter the number of brain mast cell-specific nuclei in the thalami of male rats and thus can provide targeted delivery of neuromodulators to specific regions of the brain that process information concerning the normal physiological state of the animal.

The role of serine proteases and serine protease inhibitors in the migration of gonadotropin-releasing hormone neurons.

BACKGROUND: Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH) neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes. RESULTS: Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1), and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction. CONCLUSIONS: These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.

HUMAN MIDSIZED NEUROFILAMENT EXPRESSION IN TRANSGENIC MOUSE-DERIVED GRAFTS FACILITATES STUDY OF GRAFT-HOST INTERACTIONS IN HYPOGONADAL MICE.

Our previous studies established that targeted axonal outgrowth into the host median eminence (ME) from grafted gonadotropin-releasing hormone (GnRH) neurons is essential for stimulation of reproductive function in hypogonadal (HPG) mice homozygous for a deletion in the GnRH gene. In the current experiments transgenic mice expressing the human midsized neurofilament NF(M) were used as sources of grafts to clarify the extent of transplant-derived innervation of the host that accompanies this dramatic recovery process. Preoptic area (POA) tissue from 1- or 2-dayold transgenic pups was implanted in the third ventricle of adult male HPG mice. Polymerase chain reaction (PCR) analysis verified that each donor bore the transgene. Immunohistochemistry using a monoclonal antibody specific for human NF(M) revealed intensely immunoreactive parvicellular soma and proximal dendrites in the grafts. Interestingly, human NF(M)-positive neurons also migrated out of the graft into the host hypothalamus. In the animal with the most dramatic increase in testicular and seminal vesicle weight, human NF(M)-positive cells were observed within the host ME. In some cases, human NF(M)-positive axonal bundles were present within the wall of the third ventricle of the host as well as in the host ME. These were GnRH negative. More extensive anatomical studies will delineate the degree and patterning of axonal outgrowth. In these early studies, however, it is apparent that neuronal fiber outgrowth into the host brain is not exclusive to GnRH fibers from the preoptic area grafts. This is important information in regard to our studies of the specificity of the signals derived from the ME that may be involved in GnRH fiber targeting.

FUNCTIONAL ASSESSMENT OF INTRAHYPOTHALAMIC IMPLANTS OF IMMORTALIZED GONADOTROPIN-RELEASING HORMONE-SECRETING CELLS IN FEMALE HYPOGONADAL MICE.

The hypogonadal (HPG) mouse is a mutant that lacks a functional gonadotropin-releasing hormone (GnRH) gene. In this study, female HPG mice received bilateral intrahypothalamic implants of an immortalized GnRH-secreting cell line (GT1-7). Nine mice were tested 42- 65 days after implantation to determine whether these cells could support spontaneous and/or N/-methyl-d,l-aspartic acid (NMDA)-stimulated luteinizing hormone (LH) secretion. When sampled via intravenous catheters, four mice had measurable LH secretion. Three of these mice responded to NMDA challenges with significant increases in circulating LH. GnRH immunocytochemistry revealed that GT1-7 cells were present in these four mice and three others in which LH values were not detectable. There were about 1200 GnRH cells dispersed within the piriform cortex and olfactory tubercle, and no tumor found in one of the HPG mice that responded to NMDA, whereas the other NMDA responders had large bilateral hypothalamic tumors. The presence or absence of such tumors did not predict the capacity to respond to the NMDA challenge with alterations in LH secretion. This study provides the first evidence that intrahypothalamic GT1-7 cells can support LH release in the HPG mouse, and that this secretion can be modified by pharmacological agents.