Effects of a Preincisional 14 Day Course of Valerian on Natural Killer Cell Activity in Sprague-Dawley Male Rats Undergoing Abdominal Surgery Essay Example

Effects of a Preincisional 14 Day Course of Valerian on Natural Killer Cell Activity in Sprague Effects of a Preincisional 14 Day Course of Valerian on Natural Killer Cell Activity in Sprague-Dawley Male Rats Undergoing Abdominal Surgery Essay Effects of a Preincisional 14 Day Course of Valerian on Natural Killer Cell Activity in Sprague-Dawley Male Rats Undergoing Abdominal Surgery Essay Effects of a Preincisional 14 day course of Valerian on Natural Killer Cell Activity in Sprague-Dawley Male Rats Undergoing Abdominal Surgery Authors: COL (ret) Norma Lynn Garrett, CRNA, PhD, USA, AN Fort Sam Houston, Texas LTC (ret) Nathaniel M. Apatov, CRNA, PhD, USA, AN Tripler Army Medical Center, HI CPT Robert Fredregill, SRNA, BSN, USA, AN Tripler Army Medical Center, HI CPT Aaron Johnston, BSN, USA, AN Tripler Army Medical Center, HI CPT John Buen, SRNA, BSN, USA, AN USUHS, Graduate School of Nursing, Nurse Anesthesia Program, Bethesda, Maryland CPT Michael Neill, SRNA, BSN, USA, AN Tripler Army Medical Center, HI Patricia S. Dixon, MS Affiliation: U.S. Army/University of Texas Health Science Center at Houston Graduate Program in Anesthesia Nursing, Fort Sam Houston, Texas Introduction: The American Cancer Society estimated that over one million new cancer cases were diagnosed in 2005 and a majority of these patients died from metastatic spread.1 The standard for treating solid tumor cancer is surgical resection. However, it has been suggested that surgical resection may in fact promote metastasis.2 One of the bodyâ„ ¢s natural defenses to combat metastasis is the activity of natural killer (NK) cells. NK cells serve as a vital mediator of detection during the early innate immune response and destruction of aberrant cells. It has been demonstrated that benzodiazepines may ameliorate surgery-induced suppression of natural killer cell activity.3 We examined the effect of a 14-day course of valerian, an herbal anxiolytic, on natural killer cell activity in Sprague-Dawley rats. Methods: Thirty-five rats were assigned to one of three groups: 1) surgical animals administered research grade valerian, 15 mg/kg solubilized in peanut oil 2) surgical animals administered peanut oil (vehicle) and 3) anesthesia only animals administered valerian. One day prior to the 14 day course of valerian, blood was drawn to assay baseline NK cell activity. On experimental day, all animals were administered isoflurane anesthesia. Surgical animals underwent a standard laparotomy whereas anesthesia only rats were anesthetized for the same period of time as the surgical rats. Twenty-four hours post-experiment animals underwent a second blood draw to assay NK cell activity. Results: Analysis of covariance (ANCOVA) was used to analyze NK cell activity (measured in lytic units). Our results suggested that there was no difference (p = 0.9) in suppression within or between groups. Conclusions: Clinical studies with valerian have been published but with small numbers and some ambiguity. Further research regarding valerianâ„ ¢s effectiveness as a modulator of NK cell activity and whether dosage or route of administration is a factor in modulation is still warranted. Keywords:?   Valerian, Natural Killer Cells, Cancer Funding: 59th Medical Wing Clinical Research Squadron, 2200 Bergquist Drive, Bldg 4430, Lackland AFB, TX 78236-5300. OBJECTIVE / HYPOTHESIS: The purpose of this study was to determine the effects of a presurgical 14-day course of valerian on Natural Killer cells activity in male Sprague-Dawley rats undergoing abdominal surgery. Our research was directed by the following research question: Would a presurgical 14-day course of valerian inhibit 24-hour postoperative surgical suppression of NK cell activity when compared to a 14-day course of vehicle in male Sprague-Dawley rats undergoing abdominal surgery Background: Cancer is ranked as the second leading cause of death in the United States and the majority of cancer related deaths have a metastatic component.4 Currently, the most common treatment for solid tumor carcinoma is surgical resection. Surgical resection results in a release of growth factors that has been suggested to promote metastasis of the remaining cancer cells.5 Surgical insult, postoperative pain, and anxiety may serve as inhibitors to successful surgical treatment by suppressing the endogenous cell mediated immune (CMI) response. CMI limits metastasis of cancerous cells by activating Natural Killer (NK) cells. NK cells are endogenous specialized large granular lymphocytes that detect and destroy aberrant cells.6 Aberrant cells lack or possess a deficit of functional major histocompatibility complex class I (MHCI) surface proteins. NK cells serve as vital mediators of aberrant cellular detection during the early innate immune response.7 Surgically induced suppression of NK cell activity can be attenuated by ameliorating the surgical stress response. In 1999, Ben-Eliyahu and colleagues examined the effects of a postoperative beta-adrenergic antagonist on Fisher 344 rats using a lung tumor retention assay (MADB106). The beta-adrenergic antagonist resulted in the inhibition of lung tumor colonization.8 In 2002, Page and colleagues examined lung tumor susceptibility in Fisher 344 rats and observed that a non-steroidal anti-inflammatory drug (NSAID) attenuated the suppression of NK cell activity caused by surgical stress. The findings from these studies compellingly suggest that surgery contributes to the suppression of the immune system, specifically, NK cell activity. There exist both physiological and psychological mechanisms responsible for the suppression of NK cell activity in the surgical patient. Physiological and psychological mechanisms related to anxiety are implicated in the modulation of the CMI. Anxiety activates the sympathoadrenomedullary systems and the hypothalamic-pituitary- adrenal axis (HPA). Neurosignals are processed in the hypothalamus, which cause the release of various neurotransmitters (e.g. norepinephrine), neuropeptides (e.g. corticotrophin-releasing factor), neurohormones (e.g. adrenocorticotropin hormone) and adrenal hormones (e.g. catecholamines and corticosteroids). Both catecholamines and corticosteroids are suggested to suppress the immune system and NK cell activity.9 10 Evidence from both animal and human studies suggest that anxiety is a major cause of CMI suppression. Anxiolytic medications may attenuate the negative psychological impact associated with surgical stress.11 Benaroya-Milshtein and colleagues demonstrated an increase in NK cell activity by reducing anxiety in mice.12 A 2001 study by Koga and colleagues demonstrated that anxiety suppressed NK cell activity in 144 human patients scheduled for oral surgery.13 A study by Nunez and colleagues demonstrated that benzodiazepines, such as alprazolam, could reverse the adverse effects of stress and anxiety on CMI in Sprague-Dawley rats. 14 Finally, Freire-Garabal and colleagues suggested that chronic alprazolam administration caused a dose-dependent reduction in surgically mediated stress-induced suppression of NK cell activity in mice.15 Catacholamine and cortisol levels were reduced and enhanced protection of NK cell activity in the groups receiving pre and post surgical administrat ion of alprazolam was noted. Valerian, an herbal medication with reputed anxiolytic effects, demonstrates a dose-dependant GABAergic effects in in vitro studies of rat brainstem preparations.16 Yuan and colleagues demonstrated the site of action of a known benzodiazepine and valerian to be the GABAA receptor.17 Further, Yuan and colleagues observed both direct and indirect modulation of the GABAA receptor activity but were unable to isolate the active pharmacological agents in valerian preparations. In a related study, Ortiz and colleagues demonstrated the direct and indirect modulation of the GABAA receptor utilizing oral valerian extracts in in-vitro studies of Sprague-Dawley rats.18 Human studies suggest that the effective oral dose for anxiolytic and hypnotic effects range from 400 to 900 mg administered daily.19 20 Additional research suggests that chronic administration of valerian over a minimum period of 2 weeks is necessary to achieve pharmacologically effective serum levels.21 Acute administratio n of valerian has been proposed to provide anxiolytic effects but may be related to a placebo effect. Given that NK cells play an integral role in the suppression of metastatic cancer and that anxiety suppresses NK cell activity, interventions that reduce anxiety may lead to better outcomes in patients undergoing surgical resection of solid tumor carcinomas. If perioperative anxiety can be attenuated with valerian, then surgical suppression of NK cell activity may also be attenuated. Methods A sample of 35 male Sprague-Dawley rats was partitioned into three groups with two rats utilized for model development. The rats were separated into the following groups: Valerian and surgery (11), Vehicle and surgery, the control group (11), and Valerian and anesthesia (11). The anesthesia only group assessed the effects of valerian on NK cell activity under non-surgical conditions compared to surgical conditions. For each of the groups listed above, mature male Sprague-Dawley rats; weighing 225-250 grams were used. Animals were brought in and acclimatized to the vivarium for 3-days prior to habituation and handling. Rats were housed in plastic cages on a 12-hour light/dark cycle. Animals had continuous access to food and water except for the 8-hours prior to the experiments, when only water was available. The rats were partitioned into three equal groups. We randomized the order in which the rats would be tested to ameliorate any effect that a surgeonâ„ ¢s ability might ha ve upon the outcome. For each of the 14 days prior to the experiment, animals were administered either vehicle (peanut oil) or valerian (15mg/kg), depending on the group assignment. The dose was based on a clinical trial by Cropley (2002) whose findings suggested that a 9mg/kg dose of valerian administered over 7 days was effective in reducing stress and on an rodent study suggesting that a single dose at 30 mg/kg significantly prolonged emergence time from anesthesia.22 One day prior to the experiment, animals were briefly anesthetized with isoflurane to withdraw blood via a cardiac puncture for baseline NK cell assay. On the experiment day, surgical animals underwent a standard laparotomy with isoflurane anesthesia; anesthesia only animals received isoflurane at the same time and dose as the surgery animals. Once anesthetized, all animals were injected intramuscularly (IM) with penicillin (25,000 units/kg), and the abdomen of the surgery animals was shaved and prepared with betadine. Surgery consist ed of a 4 cm midline incision through the skin and abdominal muscle wall followed by the externalization of a 10 cm segment of the small intestine for a period of 4-minutes. During the first minute, the intestine was gently rubbed between two pieces of gauze in 4 locations as a standard irritant; this procedure promoted the release of local inflammatory factors. For the remainder of the 4-minute period, the intestine was covered with saline-soaked gauze to maintain the moisture content. The intestines were then returned to abdominal cavity, irrigated with saline, and the muscle and skin layers were stapled. Isoflurane gas was discontinued after stapling was completed. At 24 hours after surgery, all animals were anesthetized for the withdrawal of approximately 2 ml of blood via cardiac puncture for the post surgical NK cell assay. Exactly 1 ml was placed in a heparinized tube (20 U/ml) with 3.0 ml PBS with bovine serum albumin (BSA, 1 gm/L) and centrifuged at 500 g for 10 minutes. The supernatant was then aspirated down to the original blood volume. The blood was washed two times with 3.0 ml medium, using the same centrifugation and aspiration procedure. YAC-1 cells, a standard cell line for the assessment of rodent NK cell activity, are maintained in the above-specified complete medium. To radiolabel the cytoplasm of the YAC-1 cells, approximately 15(106 cells are incubated with 0.2 mCi of 51NaCrO4, 150 (l medium, and 200 (l FCS for 90 minutes. The medium used for this assay is the same as the above-described medium, except that the FCS content is 15% by volume. After incubation, target cells are washed 3 times in medium and their concentra tion adjusted to 8(105 cells per ml (approximately 6:1 leukocytes:YAC-1) for the lowest effector:target (E:T) ratio. Because whole blood is used, it is necessary to serially dilute the target cells to achieve the various E:T ratios. For each E:T ratio, 100 (l of washed blood was aliquoted into a well of a microtiter plate, and 150 (l of 51Cr-labeled YAC-1 target cells in medium was then added on top of the blood. Plates were centrifuged at 500g for 10 minutes, which creates a buffy layer of leukocytes and target cells on top of the red blood cells. Plates were then incubated for 4 hours, centrifuged at 500 g for 10 minutes, and 100 (l supernatant was harvested for determination of 51Cr release in the gamma counter. The spontaneous release and maximum release (incubation of YAC-1 cells in medium and 1 Normal HCl, respectively) was measured for each tumor cell concentration separately, and percent specific lysis was calculated using the formula: [(Experimental Spontaneous) ( (Maximum Spontaneous)] (100. Data Analysis: A total of 6 subjects were omitted from the various groups. Two subjects (one from each surgical study group) were omitted due to post operative wound dehiscence. Four subjects were omitted due to post-feeding pulmonary edema. As expected, all groups had significantly suppressed NK cell activity compared to pre-experimental values (p