Michael F. Callahan

Stress increases oxytocin release within the hypothalamic paraventricular nucleus

Evidence indicates that the hypothalamic paraventricular nucleus (PVN) and oxytocin (OT) neurons in particular play a role in the physiological response to stress. Microdialysis (MD) experiments were performed to determine whether OT is released into the PVN during shaker stress. Male rats were prepared with venous catheters and PVN guide cannulae. OT and vasopressin (VP) release into PVN and peripheral blood were measured under basal conditions and during and after shaker stress (10 min at 110 cycles/min). Stress produced a specific increase in PVN and plasma OT. Dialysate OT levels were 0.3+/-0.1, 2.8+/-1.2 and 1.3+/-0.6 pg/sample (control, stress and recovery, respectively). Plasma OT was significantly increased during stress (3.7+/-1.2 vs. 11.7+/-2.3 pg/ml, basal vs. stress, respectively). When MD probes were located outside the PVN, there was no increase in OT release, demonstrating site specificity. Stress produced no change in VP levels, either in dialysate or plasma. These results show that OT, but not VP, is released into the PVN and peripheral blood in response to shaker stress. The data raise the possibility that local release of OT into the PVN plays a role in the neuroendocrine stress cascade.

Systemic osmotic stimulation increases vasopressin and oxytocin release within the supraoptic nucleus

Vasopressin (VP) and oxytocin (OT) are released within the hypothalamic nuclear region in response to direct microdialysis with hypertonic solutions. Experiments were performed to determine whether systemic osmotic stimulation causes changes in intranuclear peptide release within the supraoptic nucleus (SON). A hypertonic sodium chloride solution was injected intraperitoneally (ip) or intravenously (iv) and microdialysis techniques were used to simultaneously monitor central and peripheral peptide release in urethane anesthetized rats. Systemic osmotic stimuli elicited increases in intranuclear peptide release which were delayed and long‐lasting, occurring over a 2.5 h period. In contrast, plasma peptide levels peaked at 30‐min after the stimulus. The results demonstrate that increased plasma sodium elicits an increase in VP and OT release into the extracellular space of the hypothalamic SON. The different patterns of peptide release in plasma and brain point toward the possibility of independently regulated release into the different compartments.

EFFECT OF LOCALLY DELIVERED IGF-1 ON NERVE REGENERATION DURING AGING: AN EXPERIMENTAL STUDY IN RATS

Age is an important predictor of neuromuscular recovery after peripheral nerve injury. Insulin‐like growth factor 1 (IGF‐1) is a potent neurotrophic factor that is known to decline with increasing age. The purpose of this study was to determine if locally delivered IGF‐1 would improve nerve regeneration and neuromuscular recovery in aged animals. Young and aged rats underwent nerve transection and repair with either saline or IGF‐1 continuously delivered to the site of the nerve repair. After 3 months, nerve regeneration and neuromuscular junction morphology were assessed. In both young and aged animals, IGF‐1 significantly improved axon number, diameter, and density. IGF‐1 also significantly increased myelination and Schwann cell activity and preserved the morphology of the postsynaptic neuromuscular junction (NMJ). These results show that aged regenerating nerve is sensitive to IGF‐1 treatment. Muscle Nerve, 2009

Disease progression and phasic changes in gene expression in a mouse model of osteoarthritis.

Osteoarthritis (OA) is the most common form of arthritis and has multiple risk factors including joint injury. The purpose of this study was to characterize the histologic development of OA in a mouse model where OA is induced by destabilization of the medial meniscus (DMM model) and to identify genes regulated during different stages of the disease, using RNA isolated from the joint “organ” and analyzed using microarrays. Histologic changes seen in OA, including articular cartilage lesions and osteophytes, were present in the medial tibial plateaus of the DMM knees beginning at the earliest (2 week) time point and became progressively more severe by 16 weeks. 427 probe sets (371 genes) from the microarrays passed consistency and significance filters. There was an initial up-regulation at 2 and 4 weeks of genes involved in morphogenesis, differentiation, and development, including growth factor and matrix genes, as well as transcription factors including Atf2, Creb3l1, and Erg. Most genes were off or down-regulated at 8 weeks with the most highly down-regulated genes involved in cell division and the cytoskeleton. Gene expression increased at 16 weeks, in particular extracellular matrix genes including Prelp, Col3a1 and fibromodulin. Immunostaining revealed the presence of these three proteins in cartilage and soft tissues including ligaments as well as in the fibrocartilage covering osteophytes. The results support a phasic development of OA with early matrix remodeling and transcriptional activity followed by a more quiescent period that is not maintained. This implies that the response to an OA intervention will depend on the timing of the intervention. The quiescent period at 8 weeks may be due to the maturation of the osteophytes which are thought to temporarily stabilize the joint.

Central Oxytocin Mediates Stress-Induced Tachycardia

To address the role of oxytocin in the control of cardiovascular reactivity, we examined the effect of central injection of oxytocin, vasopressin and mixed base antisense oligodeoxynucleotides on stress‐induced cardiovascular and endocrine changes. Antisense oligomers were injected into the paraventricular nucleus (PVN), 4 h prior to the stress test. The oxytocin antisense abolished the tachycardia produced by 5 min of shaker stress. The blood pressure and plasma oxytocin responses were not different between the groups. PVN levels of OT were reduced in the oxytocin antisense‐treated group while brain stem levels were increased. These results demonstrate the importance of a specific peptide system, the PVN/oxytocin axis, in stress‐induced tachycardia. Further, the data illustrate the effectiveness of short‐term treatment with antisense oligomers on physiological responses.

Neuroendocrine Effects of Dehydration in Mice Lacking the Angiotensin AT1a Receptor

Angiotensin (Ang) type 1a (AT1a) receptors are critical in the control of blood pressure and water balance. Experiments were performed to determine the influence of dehydration on brain Ang receptors and plasma vasopressin (VP) in mice lacking this receptor. Control or AT1a knockout (AT1aKO) male mice were give water ad libitum or deprived of water for 48 hours. Animals were anesthetized with halothane, blood samples were collected by heart puncture, and brains were processed for Ang-receptor autoradiography with 125I-sarthran (0.4 nmol/L). Dehydration produced an increase in AT1 receptors in the paraventricular nucleus (PVN) and anterior pituitary (AP) in control mice (PVN: 70+/-16 versus 146+/-10 fmol/mg protein; AP: 41+/-7 versus 86+/-15 fmol/mg protein). No changes were noted in the median preoptic nucleus. The majority of the brain receptors were of the AT1 subtype. There was little or no specific Ang binding in AT1aKO mice and no effect of dehydration. Plasma VP levels were elevated in the halothane-anesthetized animals (>200 pg/mL) with no significant effect of dehydration. A separate experiment was performed with decapitated mice anesthetized with pentobarbital. Dehydration increased plasma VP in control mice, from 3.3+/-0.6 to 13.3+/-4.7 pg/mL, whereas no change was noted in the AT1aKO mice, 5.1+/-0.3 versus 6.1+/-0.7 pg/mL (water versus dehydration). These results demonstrate a differential response to dehydration in mice lacking AT1a receptors. There was no evidence for AT1 receptors of any subtype in the brain regions examined and no effect of dehydration on VP secretion or brain Ang receptors.

Excitotoxin paraventricular nucleus lesions: stress and endocrine reactivity and oxytocin mRNA levels

Electrolytic lesion of the paraventricular nucleus (PVN) of the hypothalamus blocks the tachycardia response to stress. The current study examined the effects of chemical lesion of PVN parvocellular neurons on the cardiovascular and endocrine responses to stress and on the content of hypothalamic oxytocin (OT) mRNA levels. Acute footshock stress increased heart rate in both ibotenic acid lesion and control groups of animals; however, the tachycardia was significantly lower in animals with a PVN lesion than the controls. Lesion of the PVN also attenuated the increase in plasma OT induced by stress, 4-fold in the lesion group versus 20-fold for the controls. There was not a generalized decrease in hormonal responsiveness since the OT response to an osmotic challenge was exaggerated in the lesion group. There was no difference between the groups in the arterial pressure and vasopressin responses to acute stress. Neurotoxin lesions of the PVN also resulted in significant depletions of VP and OT in all levels of the spinal cord and decreased OT levels in the dorsal brainstem. Ibotenic acid lesions of the PVN resulted in no significant changes in OT mRNA in the PVN, SON and PP. In addition, the 48-h dehydration resulted in a significant increase in plasma OT and OT mRNA in the PVN. These data indicate that the parvocellular neurons of the PVN play a role in integration of cardiovascular and endocrine responses to both stressful and osmotic stimuli and provide further evidence that parvocellular OT and VP neurons project to the brainstem and spinal cord.

Effects of tetrodotoxin on osmotically stimulated central and peripheral vasopressin and oxytocin release

Tetrodotoxin (TTX) was used to (1) distinguish between axonal and dendritic/somatic release of vasopressin (VP) and oxytocin (OT) within the supraoptic nucleus (SON) and (2) to determine whether neuronal inputs trigger intranuclear peptide release in the response to osmotic stimulation. Microdialysis was used to administer TTX (10(-6) M or 10(-4)M) bilaterally into the SON with simultaneous monitoring of central and peripheral peptide release and mean arterial pressure in urethane-anesthetized male rats. Osmotic stimuli were given via the microdialysis probe (1 M NaCl-artificial CSF) or injected intraperitoneally (3.5 M NaCl; 600 mu l/100 g b.w.) SON perfusion with TTX did not alter basal intranuclear VP or OT release or the intranuclear peptide response to direct NaCl stimulation of the SON. However, TTX treatment abolished the effect of peripheral osmotic stimulation on central peptide release. Basal plasma peptide levels were significantly reduced by TTX, e.g. decreases of 94.8 and 75.8% for VP and OT, respectively. TTX also blocked the peripheral endocrine and cardiovascular responses to both modes of osmotic stimulation. The TTX insensitivity of directly stimulated intranuclear release suggests nonsynaptic peptide release from dendrites and/or cell bodies. The ability of TTX to abolish the central peptide response to systemic osmotic stimulation demonstrates that intranuclear release is a part of a cascade produced by osmotic activation of multisynaptic pathways.

Histopathology of naturally occurring and surgically induced osteoarthritis in mice.

OBJECTIVE The morphology of lesions in mouse models of osteoarthritis (OA) has not been comprehensively characterized, in part because current histological assessments of OA focus primarily on articular cartilage (AC). In the present study, sections of murine stifle joints with naturally occurring (aged animals) and surgically induced (destabilized medial meniscus, DMM) OA were examined using a newly developed histological grading scheme that includes quantitative measurements and semiquantitative grades to evaluate multiple joint tissues. DESIGN The data collected was analyzed using Principal Components Analysis (PCA); factor scores for each joint were generated. Individual parameters and factor scores were compared between surgical groups and among age groups. For comparison, the original Mankin Histological-Histochemical Grading System (HHGS) also was applied. RESULTS Overall, lesions were most severe in the medial tibial plateaus. Significant changes in AC and neighboring bone were identified in surgically induced models and in naturally occurring disease. Mean factor scores provided a comprehensive evaluation of joint changes. An important new finding was that chondrocyte cell death within the AC was a commonly identified lesion and its extent significantly increased with age. While the Mankin HHGS detected significant overall differences in OA severity between surgical groups, it was not sensitive in detecting age-related differences, nor did it provide information regarding changes in individual tissues. CONCLUSION These results demonstrate the utility of this newly developed murine OA grading scheme in identifying lesions in AC and in other joint tissues. Surgically induced changes were similar to those occurring naturally with aging.

How age impairs the response of the neuromuscular junction to nerve transection and repair: An experimental study in rats.

Age is the most important predictor of clinical outcome after peripheral nerve injury. The stability of the neuromuscular junction (NMJ) after denervation is thought to be central to neuromuscular recovery. Stability is characterized by maintenance of the motor endplate and mRNA upregulation of the constituent nicotinic acetylcholinergic receptor (nAChR) subtypes and the muscle regulatory factors (MRFs). The purpose of this study was to determine the effect of age on the recovery and stability of the postsynaptic NMJ after peripheral nerve injury. Young and aged rats underwent transection and repair of the tibial nerve. At 1, 2, 4, 8, or 16 weeks following transection, the gastrocnemius was examined for electrical recovery, NMJ fragmentation and endplate area, mRNA, and protein levels of the MRFs and nAChR subtypes. After nerve injury, aged NMJ exhibited significant fragmentation and loss of motor endplate area while the young NMJ remained relatively stable. Concomitantly, age impaired peak upregulation of the MRFs and nAChRs. However, expression of γ‐nAChR and myogenin after nerve injury was not affected by age. These data support the claim that upregulation of the nAChRs and MRFs may play an important role in maintaining NMJ stability following nerve transection and repair. Furthermore, expression of γ‐nAChR and myogenin does not appear to prevent age‐related NMJ fragmentation and loss of endplate area after nerve injury. These impairments of the aged NMJ response to injury may contribute to the poor neuromuscular recovery seen after nerve injury in this population.