Orexinergic activity modulates altered vital signs and pituitary hormone secretion in experimental sepsis

Abstract

Objectives: Sepsis is a common, lethal poorly understood disorder affecting nearly a million Americans annually. The syndrome is characterized by altered cardiodynamics, respiration, metabolism, pituitary function, arousal, and impaired interaction among organ systems. The immunologic and endocrine systems, which are in part responsible for organ-organ communication, have been studied extensively in sepsis. However, little is known about sepsis-induced changes in central nervous system activity.

Hypothesis: A defect in hypothalamic neurons secreting the neurotransmitter orexin modulates physiologic derangements in sepsis.

Design: Animal study.

Setting: University Research Laboratory.

Interventions: Male C57Bl6 mice were made septic using cecal ligation and puncture. Data were collected 24 or 48 hours later, blood was collected, animals were killed, and brain tissue was harvested, fixed, and sectioned. Hypothalamic sections were subjected to immunohistochemistry using antibodies to orexin and c-Fos, a marker of neuronal activity. In a separate cohort of mice, cannulas were placed in the right lateral cerebral ventricle. Cecal ligation and puncture was performed 1 week later. At 24 or 48 hours post-cecal ligation and puncture, vital signs were measured, and1 µL of saline with or without 3 nmol orexin-A was infused. Vital signs were repeated at 25 or 49 hours post-cecal ligation and puncture, blood was collected, animals were killed, and brains were removed, fixed, sectioned, and stained.

Measurements and main results: Orexinergic activity decreased six-fold following cecal ligation and puncture. This change was associated with decreases in arousal, temperature, and heart and respiratory rates. Levels of selected pituitary hormones increased 24 hours post-cecal ligation and puncture but were significantly lower than baseline at 48 hours. Injection of orexin-A increased vital signs to baseline levels. Hormone levels were unaffected at 25 hours but increased to supranormal levels at 49 hours.

Conclusions: Sepsis-induced changes in activity, vital signs, and pituitary hormones are modulated by the orexinergic system. This finding implicates central nervous system dysfunction in the pathogenesis of sepsis, suggesting further study of neurological dysfunction to identify novel approaches to management.

Figure 1.

Effects of cecal ligation and puncture–sepsis on orexinergic activity. Representative sections stained for orexin and c-Fos. Tissue harvested from four surviving mice 48 hr after the induction of sepsis via cecal ligation and two 23-gauge punctures or from four unoperated or four sham-operated controls. Orexin-containing neurons (fluorescent green), c-Fos containing neurons (fluorescent red). Green arrows indicate orexin-positive, and c-Fos-negative neurons. Yellow arrow depicts orexin-negative, and c-Fos-positive neurons. White arrows indicate neurons positive for both c-Fos and orexin. A, Section from unoperated control. B, Section of tissue harvested 48 hr after sham operation. C, Section of tissue harvested 48 hr after 2CLP. CLP = cecal ligation and puncture.

Figure 2.

Changes in physiologic variables in awake mice. White bars = TO control; cross-hatched bars = value prior to saline injection into lateral ventricle; light gray bars = value prior to orexin injection; dark gray bars = value 1 hr after saline injection; black bars = value 1 hr after orexin injection; n = 4 for data depicted on each bar. Data are mean ± sd. X-axis on all graphs: time following sham operation/2CLP. Statistical significance was determined using two-way (time, SO/CLP) analysis of variance. Multiple comparisons were assessed using Sidak test. A, Temperature (Temp). Y-axis: Temp in degrees Celcius. Analysis of variance (ANOVA): time, p < 0.0001; treatment (saline/orexin), p = 0.0087; interaction, p < 0.0001. B, Heart rate (HR). Y-axis: beats/min. ANOVA: time, p < 0.0001; treatment (saline/orexin), p < 0.0027; interaction, p <0.0001. C, Respiratory rate (RR). Y-axis: breaths/min. ANOVA: time, p < 0.0001; treatment (saline/orexin), p = 0.0037; interaction, p = 0.0002. * = Significantly different than value at T0. † = Significantly different than value prior to injection. ^ = Significantly different than value at same time point following saline injection. SO = sham operation, CLP = cecal ligation and puncture.

Figure 3.

Changes in pituitary hormones in awake mice. White bars = T0 control, gray bars = value 1 hr after injection of saline, black bars = value 1 hr after injection of orexin. Data are mean ± sd; n = at least four for data depicted on each bar. X-axis on all graphs: time following sham operation/2cecal ligation and puncture. A, Adrenocorticotrophic hormone (ACTH). Y-axis; pg/mL. Analysis of variance (ANOVA): time, p < 0.0001; treatment (saline/ orexin), p < 0.0001; interaction, p < 0.0001. B, Thyroid stimulating hormone (TSH). Y-axis: ng/mL. ANOVA: time, p = 0.034; treatment (saline/orexin), p < 0.0001; interaction, p < 0.0001. C, Growth hormone (GH). Y-axis: ng/mL. ANOVA: time, p = 0.0073; treatment (saline/orexin), p < 0.0001; interaction, p < 0.0001. D, Prolactin (Pro). Y-axis: ng/mL. ANOVA: time, p = 0.0019; treatment (saline/orexin), p < 0.0001; interaction, p < 0.0001. (E) Follicle stimulating hormone (FSH). Y-axis: ng/mL. ANOVA: time, p < 0.0001; treatment (saline/orexin), p < 0.0001; interaction, p < 0.0001. ANOVA: time, p = 0.011; treatment (saline/orexin), p < 0.0001; interaction, p < 0.0001. * = Significantly different than value at T0. † = Significantly different than uninjected value. ^ = Significantly different than value at same time point following saline injection. § = Significantly different than value following injection of saline at 24 hr.