Chronic THC Administration to Adolescent Rats Has Some Benefits for Skeletal Development 1 Cash, C N; 2Mary, M N; 1Smith R P; 2Boyle, P; 3Volkow, N D; 4Wang, G J; 4Robinson, L; 4Ananth, M; 5Wigal, T; 5Swanson, J M; 4Thanos, P K; +2Komatsu, D E 1 2 3 University of Tennessee, Memphis, TN, Memphis, TN, Laboratory of Neuroimaging, NIAAA, NIH Bethesda, MD, 4Behavior Neuropharmacology and Neuroimaging Lab, Medical Dept. Brookhaven National Laboratory, Upton, NY, 5Child Development Center, University California Irvine, Irvine, CA Senior author [email protected] METHODS: Animals – Forty-eight male, 4 week-old Sprague Dawley rats (Harlan, Indianapolis, IN) were randomly divided into 6 treatment groups (N=8). Four of the groups were administered THC daily by IP injection. Two different doses, low (0.75 mg/kg) and high (2mg/kg) were used. Two vehicle control groups were given saline injections. Three groups (Vehicle, Low, High) were treated for 21 days and then immediately sacrificed (Standard). The other three groups were similarly treated for 21 days and then allowed to recover for 15 days with no treatment prior to sacrifice (Recovery). Following sacrifice, vertebra, tibia, and femora were removed for testing. All animal procedures were approved by the Brookhaven National Laboratories IACUC and met or exceeded all relevant guidelines for the humane use of animals in research. Measurements/DXA Scans – The length and diameter (A-P and M-L) of each left femur, right tibia, and L5 vertebra was first measured using digital calipers (Mitutoyo, Aurora, IN). Subsequently, they were scanned using a small animal dual energy X-ray absorptiometry (DXA) scanner (PixiMUS, GE Lunar, Madison, WI) to quantify bone mineral density (BMD), bone mineral content (BMC), and area. Biomechanical Testing – The biomechanical testing was performed on left femora and L5 vertebrae. The femora were subjected to 3-point bending tests to failure while the vertebrae were tested in uniaxial compression to failure. All tests were performed using an ElectroForce 3200 (Bose, Eden Prairie, MN). The femora were centered in a custom designed jig and loaded on the A-P axis at a cross-head speed of 0.1mm/sec. The vertebrae first had their proximal and distal ends cut parallel using a diamond wafer saw (Dremel, Robert Bosch, Farmington Hills, MI). They were then positioned vertically between two steel platens and loaded axially a cross-head speed of 0.1mm/sec. Following testing, Force vs. Displacement curves were plotted in Excel (Microsoft, Redmond, WA) and Ultimate Force (N), Energy to Failure (mJ), and Stiffness (N/mm) were calculated using a set of custom macros. Statistical Analysis – In order to assess differences between groups, ttests were performed using Excel. For all tests, p-values < 0.05 were considered significant. RESULTS: The results of this study identified several trends regarding the effects of chronic THC administration on the skeletal system. Analysis of the Standard treatment groups showed a few differences with some parameters in THC treated rats higher than vehicle controls and others lower. However, the Recovery animals showed a different response with potentially beneficial skeletal effects seen in THC treated animals. Specifically, the high dose group demonstrated increases in all assessed parameters, as compared to the control group (Figure 1). Figure 1 The overall trend, an initial decrease and resulting increase, in bone development is represented by this graph. The starred comparison was significant with a p-value of 0.0423. Average Femoral Area 2.5 2.4 centimeter2 2.3 * 2.2 2.1 2 1.9 Saline 0.75mg/kg 2.0 mg/kg 0.75, 15 day recovery 2.0, 15 day recovery Saline, 15 day recovery While many of these increases did not achieve significance, the recorded mean for each tested parameter in the recovery treatment groups did overwhelmingly exceed those of their vehicle counterpart. Furthermore, one statistically significant comparison of area was achieved between two of the initial groups. The low dose group demonstrated significant increases in L5 area when compared to vehicle controls (Figure 2). Figure 2 This graph shows the results from the vertebral biomechanical study. The starred comparison was significant with a p-value of 0.0484. Average Vertebral Area 0.8 0.75 centimeter2 INTRODUCTION: Among US teenagers, marijuana is currently the most widely used illicit drug. Though much research has focused on the euphoria producing cannabinoid 1 (CB1) receptor in the brain, far fewer studies have been devoted to the cannabinoid 2 (CB2) receptor present in peripheral tissues. Both CB1 and CB2 receptors are activated by tetrahydrocannabinol (THC), the main psychoactive compound present in marijuana, but the action of CB2 in peripheral tissues remains unknown. Within the skeletal system, osteoblasts and osteoclasts express the CB2 receptor. However, no consensus exists regarding how THC acts through this receptor to affect the balance between anabolic and catabolic activity. Some studies have demonstrated that THC not only up-regulates osteoblast activity, but also down-regulates osteoclast activity, suggesting that the CB2 receptor may be a viable target for skeletal anabolic therapy. This study evaluated the effects of chronic THC administration on the skeletal development of adolescent rats in order to ascertain whether or not THC affects this process. 0.7 0.65 0.6 0.55 Saline 0.75mg/kg 2.0 mg/kg 0.75, 15 day recovery 2.0, 15 day recovery Saline, 15 day recovery DISCUSSION: Having compared the skeletal effects of chronic THC administration on two separate timelines, one with and one without recovery, this study has shown that chronic THC administration has no detrimental effects on skeletal development. Furthermore, following 15 days of recovery, THC treated animals showed some evidence of superior skeletal properties compared to vehicle treated controls. These findings suggest that the potential beneficial effects of THC on skeletal accrual may require either substantial time to develop or recovery periods with no administration of THC. It is possible that stimulation of the CB2 receptor in osteoblasts initiates a slow process that only after an initial lag period results in measurable changes at the tissue level, or that a rebound response due to removal of CB2 stimulation is required for benefits to be seen. Additional studies evaluating the molecular response of bones to THC administration and different treatment timing designs will be invaluable in further elucidating these responses. In addition, studies conducted in rats that have already achieved skeletal maturity or those with osteopenia may prove valuable in fully ascertaining the effects of THC administration of skeletal integrity. Since adolescent hormone levels significantly differ from those of the elderly and the skeletal system is greatly influenced by the endocrine system, a hormonal shift might allow for a greater influence by the cannabinoid receptors. Therefore, future studies will be required before the CB2 receptor can be validated as a target for skeletal anabolic therapy and bone loss can potentially be included amongst glaucoma, wasting syndrome, and pain as a clinical indication for medicinal cannabis. Poster No. 1704 • ORS 2011 Annual Meeting
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