Christopher P. Phelps

Role and Mechanisms of Interleukin-1 in the Modulation of Neurotoxicity

Objective: Recent studies on cerebral ischemia in the rat have demonstrated that administration of interleukin-1 receptor antagonist (IL-1ra) markedly reduces the volumes of infarcts which are associated with N-methyl-D-aspartate (NMDA)-mediated neurotoxicity. These observations suggested that endogenous interleukin-1 (IL-1) may be involved in the mediation of excitotoxic neuronal injury following ischemia. Method: In the present studies, we examined the role of interleukin-1β (IL-1β) in NMDA-related and microglia-induced excitotoxicity in cocultures of mixed neurons and microglia. Results: Our observations in these mixed cultures indicated that addition of IL-1β exaggerated NMDA and glutamate-evoked hippocampal neuron death. Addition of microglia, activated by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), to cocultures of cortical neurons and glia induced significantly greater neurotoxicity when compared with cocultures of cortical neurons and untreated microglia. This neurotoxicity did not require that activated glia be in cell-to-cell contact with neurons. Addition of either IL-1ra or the NMDA receptor antagonist MK-801 to cocultures of cortical neurons and activated glia partially reversed the neuronal damage mediated by activated microglia. Finally, IL-1β concentrations in the supernatant of cocultures of cortical neurons and microglia treated by LPS and IFN-γ were markedly increased when compared with coculture of neurons with untreated microglia. Conclusion: These results suggest that both the NMDA receptor and the IL-1 receptor are involved in microglia-mediated neurotoxicity.

Effect of D-fenfluramine on neuropeptide Y concentration and release in the paraventricular nucleus of food-deprived rats

Recent evidence indicates that Neuropeptide Y (NPY) is an important signal in the hypothalamic neural circuitry that stimulates feeding in the rat. Administration of d-fenfluramine (FEN) has been shown to rapidly inhibit feeding in the rat. Because food deprivation increases the levels and release of NPY in the paraventricular nucleus (PVN) of the hypothalamus, the aim of this study was to investigate whether the rapid anorectic effects of FEN in food-deprived (FD) rats are associated with alterations in the hypothalamic NPYergic system. In the first experiment, the effect of FEN (10 mg/kg) on NPY concentrations in nine microdissected hypothalamic sites was assessed by radioimmunoassay (RIA) in rats either food deprived for 3 days or fed ad lib during the experimental period. In response to food deprivation, NPY concentrations increased significantly in the PVN and arcuate nucleus, but NPY levels remained unchanged in the remaining seven hypothalamic sites. In control rats maintained on ad lib food supply, FEN injection produced little effect on NPY concentration in hypothalamic sites. However, FEN suppressed NPY levels selectively in the PVN of FD rats, so that NPY concentrations measured in the nucleus were within the range found in satiated control rats. In the second experiment, the effect of FEN on NPY release in the PVN was examined in FD rats by the push-pull cannula (PPC) technique. NPY levels in the PPC perfusate were unchanged in FD rats during the period 30-120 min after saline or FEN injection. Also, the mean rate of NPY release was similar in vehicle- and FEN-treated FD rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytokine and adrenal axis responses to endotoxin.

The push-pull cannula (PPC) technique was applied to examine the kinetics of in vivo concentration changes in male rat brain extracellular fluid (ECF) of endogenous interleukin-1beta (IL-1beta) and corticotrophin releasing hormone (CRH) after a peripheral injection of lipopolysaccharide (LPS) (25 microg/100 g b.wt. intravenously). In addition, IL-1beta, adrenocorticotropin hormone (ACTH) and corticosterone concentrations in plasma were also measured at selected intervals after LPS challenge. Administration of LPS resulted in a progressive increase in the concentrations of IL-1beta in brain hypothalamic ECF. A significant increase from the zero time mean value of 77+/-10 to 393+/-88 pg/ml at the 15-min interval was recorded. The increase in IL-1beta concentration in hypothalamic ECF reached a peak of 883+/-237 pg/ml at 30 min post-LPS. CRH concentration in the same hypothalamic ECF was 41+/-17 pg/ml at time zero, 97+/-15 pg/ml at 15 min and at 30 min was significantly increased (215+/-56 pg/ml). A time course of significant increases at 30 min in plasma concentrations of IL-1beta, ACTH and corticosterone was also recorded in the same animals described above. The data show that a peripherally administered LPS bolus elicited an early (over 15 min post-injection) increase in brain ECF IL-1beta concentration; additional significant increases in hormones released from the hypothalamic-pituitary-adrenal (HPA) axis were recorded at 30 min post-LPS injection. These observations support the concept of an early change in hypothalamic ECF concentration of IL-1beta preceding LPS-induced activation of the HPA axis.

Sexual behavior and neural degeneration following hypothalamic knife cuts

Frontal (FC), frontal lateral (FLC) or sham deafferentations were performed on adult ovariectomized (OVX) female rats. Cuts were produced retrochiasmatically with a modified Halasz knife equipped with an extrudable blade (1.5 mm radius). At 60 days after surgery each animal was tested for lordosis behavior (LQ) following either 3 daily injections of estradiol benzoate (EB) alone or with progesterone (P). Male sex behavior was tested 5 times at 4 day intervals during a 21 day regime of daily treatment with testosterone propionate. Additional FC and FLC rats were sacrificed at 10 and 14 days after surgery and degeneration patterns were studied using the Fink-Heimer method. Brains were removed and prepared for histological examination of cut location and dimensions in each rat. Behavioral effects of knife cuts were analyzed by 2 methods: (1) grouping based on common areas of glial scars; (2) a multiple regression analysis of cut morphometrics with sex behavior. No group differences were found when LQ of FC and FLC rats were compared with Sham animals after EB alone. The expected increase in LQ which follows EB and P treatment was not observed in FLC rats when means were compared. In male sex behavior tests FC and FLC groups showed more total mounts than Sham rats, however, the differences were not statistically significant. Multiple regression analysis of individual cut location and width with behavior indicated that more anterior and lateral the cut location (FC and FLC) the greater the decrement in female sex behavior. Fink-Heimer studies revealed that both FC and FLC cuts produced degeneration bilaterally in the stria medullares, habenula, fasciculus retroflexus, in addition to arcuate, ventromedial and periventricular hypothalamic nuclei. FLC cuts also resulted in bilateral degeneration in the zona incerta, the cerebral peduncles and in the medial forebrain bundle. Collectively these data indicate the complete interruption of anterior and lateral connections of the anterior hypothalamic area and mediobasal hypothalamus with the remainder of the brain in the female rat reduced lordosis behavior.

In vivo pulsatile LHRH release into the anterior pituitary of the male rat : effects of castration

The effects of castration on the concentration of luteinizing hormone releasing hormone (LHRH) in the anterior pituitary (PIT) was studied in freely-behaving male rats using a push-pull cannula for sampling. In over 70 perfusions of the PIT of rats sampled before and at multiple days after castration there was no consistent change detected in the overall amount, secretory pulse amplitude or frequency or the LHRH signal reaching the PIT.

Changes in estrogen and progestin receptor binding resulting from retrochiasmatic knife cuts

Retrochiasmatic frontolateral knife cuts (FLC) or sham operations (Sham) were performed with a Halasz-type knife. All animals were primed with estrogen plus 0.5 mg progesterone (P) and tested for lordosis both before and after surgery. Two weeks after the last test they received estradiol (E2) in Silastic capsules and were sacrificed 2 days later for determination of either nuclear estrogen receptors or cytosol progestin receptor binding in brain and pituitary (PIT). Rats which had received FLC showed significantly lower lordosis quotients relative to Shams, and relative to their own pre-surgery scores. Nuclear E2-receptor binding was significantly reduced in the hypothalamus (HYPO) following FLC, but not in preoptic area (POA) or PIT. No changes in cytosol P-receptor binding were observed in HYPO, POA or PIT following FLC. Our results suggest a positive correlation between the number of hypothalamic E2-receptors and the capacity to display lordosis, and emphasize the importance of anterolateral connections to the HYPO for the progesterone-induced facilitation of lordosis.

In vivo release of interleukin-1β into hypothalamic extracellular fluid in rats: effects of repeated sampling

Interleukin-1 beta (Il-1 beta) concentrations in extracellular fluid (ECF) withdrawn at 10-min intervals through a push-pull cannula (PPC) located in the hypothalamus were studied in freely behaving male rats for 1 h at 24 and 72 h and again at 7 days after PPC implantation. Il-1 beta concentrations in ECF were similar in the latter. However, when ECF was sampled at 3 h and again 7 days after PPC implantation, Il-1 beta concentrations were greatly elevated at 7 days when compared to all other intervals. These results demonstrate how the relationships between Il-1 beta measured in ECF and the conditions of measurement appear to be integral parts of a whole intracerebral system: cytokine concentrations appear to be inextricably bound to intrahypothalamic conditions created by the sampling device presence and frequency of use.

Brain Response to Endotoxin

Abstract The cellular and systemic responses of the brain to endotoxin [lipopolysaccharide (LPS)] administration are considered in relation to how the central nervous system receives information about the presence of LPS in blood and extracellular fluids (ECFs). In an attempt to provide an answer to the question of “How does the brain know the body is sick?” we review LPS-induced signaling pathways, reaction of the gastrointestinal (GI) tract epithelium and related vagal sensory nerves, related endothelium of local vascular beds in both the GI tract and the brain, as well as the specialized brain vasculature of circumventricular organs (CVOs) and blood vessels in the brain parenchyma. The featured signal pathways activated by low (subseptic) doses of LPS involve cytokines and prostanoids conveying messages to the brain via perivascular and blood vessel component cells and vagal visceral afferents. Activation of the hypothalamic–pituitary–adrenal (HPA) axis, as it represents a key component of the acute-phase reaction to LPS, is the functional endpoint emphasized in the studies presented. Individual animals were instrumented to permit rapid sampling from ECF in the anterior hypothalamus and concomitant peripheral blood sampling at 15-min intervals before and after intravenous LPS administration. Changes in interleukin (IL)-1β and hormones from pituitary [adrenocorticotropin (ACTH)] and adrenal (corticosterone) components of the HPA axis were studied in individual animals. Intrahypothalamic changes in IL-1β and corticotropin-releasing hormone (CRH) within the first half hour after LPS administration implicate hypothalamic mechanisms contributing to HPA axis activation after endotoxin. The importance of hypothalamus in this acute-phase reaction is examined further in another set of experiments involving excitotoxic chemical lesions of cell bodies in the anterior hypothalamic area (AHA) prior to LPS exposure. When viewed together, the results of these studies emphasize the importance of rapid communication of the periphery with the AHA via multiple routes after peripheral exposure to LPS, which allows the activation of all levels of the HPA axis during the acute-phase reaction.

Reduced ovarian function and lordosis behavior following hypothalamic knife cuts

Lordosis behavior (LQ) estrous cycles and ovarian status were studied in the same animals following either frontal (FC), frontolateral (FLC) or sham (S) surgical interruption of medial basal hypothalamic (MBH) neural connections made with a modified Halász knife (1.5 mm radius). LQ was studied in adult female rats at 7, 30, 105 and 180 day after FC, FLC or S and estrous cycles were monitored for 28 day after cuts. Vaginal cytology examined before and after S surgery indicated regular 4-5 day cycles, whereas after FC vaginal cytology became predominately cornified and after FLC it was variable. Ovarian weights and appearance at ovariectomy (OVX) supported cycle data: S ovaries had recent corpora lutea (CL); FC gonads were smaller with no recent CL and FLC gonads appeared atrophic. Multiple regression analysis of individual FC and FLC dimensions indicated that cut location and dimensions contributed significantly to the length of postoperative estrus periods. After OVX all animals were tested for LQ after estradiol benzoate (EB) and EB plus progesterone (P) priming. Although there were no group LQ differences after EB alone, S and FC animals showed an increase in LQ after EB plus P, whereas, FLC rats showed no change and this effect persisted for 180 day. Multiple regression analysis of FC and FLC coordinates indicated that the more posterior and lateral extending cuts were associated with lower LQ scores. Collectively, these data suggest a role for similar anterolateral MBH neural connections in (1) basal and cyclic gonadotrophin release and (2) EB plus P effects on lordosis behavior.

Neural damage following retrochiasmatic knife cuts in the rat

In order to define neuronal damage produced by sham, frontal and frontolateral hypothalamic knife cuts known to effect neuroendocrine and behavioral variables, we designed a retractable knife capable of delivering horseradish peroxidase as a cut was being produced. Damaged neurons filled with homogeneous reaction product were found in virtually all hypothalamic nuclei. A similar distribution of damage was produced by frontolateral (FLC), frontal (FC) or sham cuts--the primary differences being the greater density of damaged neurons in hypothalamic nuclei and labeling of the ventral tegmental area of Tsai in FLC and FC animals. The difference between FC and FLC animals was primarily one of degree of damage. The amount of damage produced by the sham surgery is consistent with the idea that sham surgeries produce endocrine dysfunctions intermediate between frontal cut animals and unoperated controls. It is interesting to note that a number of nuclei damaged by these surgeries provide aminergic innervation to the hypothalamus.