Japanese Journal of Physiology, 55, 265–277, 2005 Cutaneous Mechanical Stimulation Regulates Ovarian Blood Flow via Activation of Spinal and Supraspinal Reflex Pathways in Anesthetized Rats Sae UCHIDA, Fusako KAGITANI, Harumi HOTTA, Tomoko HANADA*, and Yoshihiro AIKAWA* Department of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Tokyo, 173-0015 Japan; and *Graduate School of Humanities and Sciences, Ochanomizu University, Bunkyo-ku, Tokyo, 112-0012, Japan Abstract: The reflex effects of noxious mechanical to a monophasic increase due to an increase in stimulation of a hindpaw or abdominal skin MAP. After spinal transection, stimulation of the on ovarian blood flow, and the reflex pathways left abdomen produced a moderate increase involved in those responses were examined in in MAP, a remarkable increase in ovarian sym- anesthetized rats. Blood flow in the left ovary was pathetic nerve activity and a slight decrease in measured using a laser Doppler flowmeter, and ovarian blood flow during the stimulation. In con- the activity of the left ovarian sympathetic nerve trast, stimulation of the right abdomen produced and mean arterial pressure (MAP) of the common a smaller response in ovarian sympathetic nerve carotid artery were recorded. Stimulation of the activity during the stimulation while it increased left or right hindpaw for 30 s produced marked the MAP to a similar degree. Ovarian blood flow increases in ovarian sympathetic nerve activity slightly increased after the end of stimulation, and MAP. Ovarian blood flow slightly decreased which was explained as passive vasodilation due during the stimulation and then slightly increased to the increase in MAP. In conclusion, stimulation after the stimulation. After the left ovarian sym- of somatic afferents affects ovarian blood flow by pathetic nerves were severed, the same stimulus inducing changes in ovarian sympathetic nerve produced a remarkable monophasic increase in activities and blood pressure. When stimulation ovarian blood flow that was explained by passive was applied to a hindpaw whose segment of af- vasodilation due to a marked increase in MAP. ferent input is far from the segment of the ovarian After spinal transection at the third thoracic (T3) sympathetic nerves, it took a supraspinal reflex level, the responses of MAP, ovarian sympathetic pathway. However, when stimulation was applied nerve activity, and ovarian blood flow to hind- to the abdomen whose spinal segment of the af- paw stimulation were nearly abolished. Stimula- ferent is close to the segment of the ovarian sym- tion of the abdomen at the right or left side for pathetic nerve output, there are spinal segmental 30 s produced slight increases in ovarian sym- reflex pathways. The present results demonstrate pathetic nerve activity and MAP. Ovarian blood that spinal reflexes depend on the laterality of the flow slightly decreased during the stimulation and stimulus, while supraspinal reflexes do not de- then slightly increased after the stimulation. After pend on the laterality of the stimulus. [The Japa- the ovarian sympathetic nerves were severed, nese Journal of Physiology 55: 265–277, 2005] the response of the ovarian blood flow changed Key words: autonomic nervous system, ovarian blood flow, ovarian sympathetic nerve, cutaneous stimulation, rat. Received on Sep 30, 2005; accepted on Oct 31, 2005; released online on Nov 1, 2005; DOI: 10.2170/jjphysiol.R2133 Correspondence should be addressed to: Sae Uchida, Department of the Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo, 173-0015 Japan. Phone: +81-3-3964-3241 (Ext. 3086), Fax: +81-3-3579-4776, E-mail: [email protected] Japanese Journal of Physiology Vol. 55, No. 5, 2005 265 S. UCHIDA et al. MATERIALS AND METHODS The ovary is innervated by autonomic nerves in addition to being under the control of hormones (see Thirty-seven virgin female Wistar rats, 4–10 months review by Burden [1]). Histological studies in rats old (body weight, 170–240 g), were used for the showed that the autonomic nerves innervating the present experiments. The estrous cycle of each animal ovary are sympathetic nerves and vagus nerves [2–5]. was determined by monitoring vaginal smears; 5 of Recently, we demonstrated in anesthetized rats that the 37 rats were in proestrus, 18 were in estrus, 2 were stimulation of the sympathetic nerve that innervates the in metestrus, and 12 were in diestrus on the day of the ovary causes a reduction in ovarian blood flow, while experiment. Rats were kept in a room with a 12 h:12 h stimulation of the vagus efferent nerve that innervates light–dark schedule, with rat chow and water provided the ovary had no effect on ovarian blood flow [6]. ad libitum. This study was approved by the Animal Furthermore, we demonstrated that application of a Committee of our institution. noxious mechanical stimulus to a hindpaw produced a Surgical procedures. Animals were reduction in ovarian blood flow during the stimulation anesthetized with urethane (1.1 g/kg, I.P.). The by activating the ovarian sympathetic vasoconstrictor trachea was cannulated and respiration was artificially nerve [6]. maintained using a respirator (Model 683, Harvard, Somatic afferent stimulation produces conscious Holliston, Massachusetts, USA). The end-tidal CO2 sensation, emotional responses, and various autonom- concentration, which was monitored by a gas monitor ic responses. These somatically-induced autonomic (1H26, NEC San-ei, Tokyo, Japan), was kept at 3–4% responses including blood flow changes are produced by controlling the respiratory volume and frequency. by activation of an autonomic efferent nerve. Some of The systemic blood pressure was continuously these autonomic responses induced by somatic stimu- recorded through a cannula in a common carotid artery lation are reflex responses called somato-autonomic with a strain gauge (TP-400T, Nihon Kohden, Tokyo, reflexes. The central reflex pathways of somato-sym- Japan). A jugular vein was cannulated for infusion pathetic reflexes consist of segmental spinal reflexes of necessary solutions. The animal was immobilized and generalized supraspinal reflexes (see reviews, [7– by administration of gallamine triethiodide (20 9]). Supraspinal reflexes seem to be activated particu- mg/kg, I.V., Sigma, St. Louis, MO). The core body larly when a limb afferent is stimulated, and segmental temperature, which was monitored in the rectum, was reflexes seem to be activated by stimulation of spinal maintained at around 37.5°C using a body temperature segmental afferents entering the spinal cord at the control system containing a thermostatically-regulated level of thoracic and higher lumbar segments. In rats, DC current heating pad and infrared lamp (ATB-1100, the sympathetic nerve innervating the ovary emerges Nihon Kohden, Tokyo, Japan). During the experiments, from the spinal cord mainly at the segments of T9 and urethane (10% of the dose used for initial anesthesia) T10 [3], while afferent inputs of the hindpaw enter the was administered I.V. every 1–2 h. spinal cord at the level of L3–L5 [10]. This anatomi- cal evidence suggests that the sympathetically-medi- ovarian laser Doppler A B aorta plexus n. flowmeter ated reduction in ovarian blood flow during hindpaw rec. right left stimulation takes supraspinal reflex pathways instead of segmental reflex pathways. ovarian The present study was performed to clarify whether artery ovary cover there are spinal and/or supraspinal reflex components uterine glass abdomen artery in the response of ovarian blood flow to cutaneous uterus stimulation. For this purpose, we applied noxious stimulation to a hindpaw whose afferent enters the spinal cord at the level of L3–L5, and also to the ab- dominal skin whose afferent enters the spinal cord at hindpaw 10 mm around the level of T9–T12 [11]. To examine the pres- Fig. 1. Schematic diagram of the experimental proce- ence of spinal reflex pathways, we prepared spinalized dures. A: Pinching was applied to the left or right hindpaw rats whose spinal cord was transected at the level of or the abdominal area (hatched area) at the left or right side T3. Furthermore, we examined the involvement of so- for 30 s. B: Blood flow in the left ovary was measured using a laser Doppler flowmeter. Efferent nerve discharges were matic afferent nerves and ovarian sympathetic efferent recorded from the left ovarian sympathetic nerve (ovarian nerves in the somatically-induced ovarian blood flow plexus nerve). Ovarian blood flow and ovarian sympathetic changes. nerve activity were recorded in different animals. 266 Japanese Journal of Physiology Vol. 55, No. 5, 2005 Fig. 1 Central Reflex Pathways of Somato-Ovarian Vascular Reflexes Measurement of ovarian blood flow. Blood monitored on an oscilloscope to guard against count- flow in the left ovary was measured in 21 rats using ing any artifacts of recordings that might arise. a laser Doppler flowmeter (ALF21D, Advance, To- In our previous study, we found that the response kyo, Japan) (Fig. 1B). After ventral exposure of the of ovarian sympathetic nerve activity to pinching of left ovary with a wide abdominal wall opening, the a hindpaw in central nervous system (CNS)–intact ovary itself was gently placed on a small plate. The rats was not influenced by cutting the bilateral vagus probe (outer diameter, 1.0 mm) of the flowmeter was nerves [6]. Therefore, in most of the experiments in gently placed in contact with a cover glass (about 7 × the present study, the vagus parasympathetic nerves 7 mm) that had been placed on a surface of the ovary were kept intact. that was devoid of any visible large vessels. Care was Severance of somatic afferent nerves. In 5 taken to avoid compressing the ovary. The probe of the rats, somatic afferent nerves innervating a hindpaw flowmeter was fixed in place using a balancing holder and abdomen were severed. The spinal nerves between (ALF-B, Advance, Tokyo, Japan).