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