Objective:

The main objective of the study was to explore the effect of Activator manipulation in OVX rats.

Methods:

Animals
The protocol, using a limited number of female Sprague-Dawley rats (6 months of age) (Charles Rivers International, Barcelona, Spain) undergoing either shamoperation (Sh) (n=10) or ovariectomy (OVX) (n=15), was approved by the Institutional Animal Care and Use Committee at the IIS-Fundación Jiménez Díaz, according to the European Union guidelines for decreasing animal pain. We complied with the 3R (‘‘replace, reduce, and refine”) experimental design recommendation aimed to reduce the number of experimental animals [21]. Rats were placed in cages under standard conditions (room temperature 20 ± 0.5ºC, relative humidity 55 ± 5% and illumination with a 12 h/12 h light/dark photoperiod), given food and water ad libitum and allowed to move without restriction.

Animal procedures
Both Sh and OVX rats were weighed and divided in two groups, respectively: not manipulated (NM) animals or those manipulated (M) using the setting 1 of the Activator V Adjusting Instrument® (Activator Methods International, Phoenix, AZ) with preload of 3.705 pounds/inch spring rate, applied onto the tibial tubercle at a 90º angle from medial to lateral side [22]. In rats of the M group (Sh =5, OVX=10), right hind limbs were adjusted with true chiropractic manipulation (TM), whereas corresponding left hind limbs were subjected to false chiropractic manipulation (FM) by firing the Activator V in the air and gently touching the tibial tubercle. These procedures were repeated 3 times/week for 6 weeks, starting once bone loss was confirmed in OVX rats (10 weeks after OVX). At the end of treatments, bone mass was determined in anesthetized (ketamine/xilacine) rats. Thereafter, animals were sacrificed by isoflurane inhalation, followed by removal of the long bones, spine and muscles (quadriceps femoris, soleus, tibialis anterior and tibialis posterior) for analysis as described below

Results:

Chiropractic manipulation improves OVX-related bone loss in rats
Although total body weight was higher in OVX rats than in Sh animals (456 ± 12 g vs 351 ± 8 g, respectively; p<0.01), bone loss occurred in the former rats as confirmed by DXA. In the long bones and vertebrae of NM-OVX rats (similar to that in FM-OVX rats when appropriate), bone mass parameters were lower than those in Sh animals (Table 1a). ActivatorV® adjustment in the tibial tubercle of the right hind limbs produced higher BMD and BMC in both the distal femur and the proximal tibia of OVX rats (TM group), even though not reaching the corresponding values in Sh rats (Table 1b). In contrast, BMD and BMC values in the proximal femur or L3-L4 vertebrae (axial bone subjected to physiological mechanical loading) were similar in both manipulated and not manipulated groups of OVX rats (Tables 2 and 3). BMD and BMC values were similar in both the long bones and vertebrae among all experimental groups of Sh rats

Chiropractic manipulation compensates in part trabecular bone alterations induced by OVX in rats
Next, we aimed to confirm whether the aforementioned improvement of bone mass was related to parallel changes in bone structure elicited by the chiropractic manipulation in OVX rats. Using CT, we evaluated several trabecular and cortical bone parameters in the long bones of the different groups of rats studied. Consistent with the observed bone mass loss in OVX rats, BV/TV and Tb.N were found to be lower, and Tb.S higher, in trabecular bone of the distal femur and the proximal tibia in both NM-OVX and FM-OVX groups of rats, compared to those in NM-Sh rats (Tables 4 and 5). It is worth noting that the TM-OVX group showed a significant improvement of these bone structure parameters (BV/TV, Tb.N and Tb.S) at both skeletal locations (Table 5). In contrast, we failed to detect any significant change in the cortical bone parameters tested (Ct. Th and M.Ar) in OVX rats, subjected or not to chiropractic manipulation (Table 5).

Chiropractic manipulation counteracts the low muscle MGF protein expression in OVX rats
We also evaluated whether chiropractic stimulus produced by the Activator V® would affect MGF production in rat skeletal muscles, related to the observed bone alterations in OVX rats. A lower MGF protein expression was observed in the quadriceps femoris, and in the tibialis anterior and posterior, but not in the soleus in NM-OVX rats in comparison to the NM-Sh group (Fig.1). This difference was counteracted by chiropractic manipulation (TM-OVX group) in both the quadriceps femoris and tibialis anterior (Fig.1). In view of these results, we decided to confirm whether TM on the right hind limbs of OVX rats could affect the FM on the contralateral hind limbs of these rats. Thus, we compared the expression of MGF in quadriceps femoris and tibialis anterior between both hind limbs in both Sh and OVX rats. We failed to observe any significant alteration in this protein expression between the left and the corresponding right muscles in both NM-Sh and NM-OVX groups, although these values of the latter group were lower than those of the former group (Fig. 2). Moreover, these values were similar in both NM-OVX and FM-OVX rats. On the other hand, higher levels of MGF protein occurred in these muscles of TM-OVX rats, compared to FM-OVX rats, confirming the local action of Activator V® (Fig.2)

Chiropractic manipulation interferes with the elevated MGF expression in the long bones of OVX rats
Considering the observed variations in MGF protein expression in the rat skeletal muscle, we proceeded to assess the possible MGF alterations which might have occurred in the rat long bones associated with Activator V® manipulation in OVX rats. We focused on osteocytes, which express MGF and are the most abundant cells in the cortical bone matrix. In both femur and tibia, MGF immunostaining in these cells was increased in OVX rats, compared to that in the NM-Sh group, but this increase was abrogated by chiropractic manipulation in these rats (Fig. 3).

Conclusion:

In conclusion, even considering the limitations of the present pre-clinical study as stated above in the Discussion, the present findings support the notion that chiropractic manipulation can improve osteoporotic bone at least in part by targeting skeletal muscle. This experimental study provides novel scientific data that open new avenues to supporting the application of chiropractic manipulation in bone loss-related situations.