Effects of 9 weeks hindlimb suspension on neuromuscular activity patterns in rat

It is well known that gravitational unloading induces muscle atrophy associated with a shift of fiber type in slow-twitch muscle. Ishihara et al. (1996 & 1997) reported that 2 weeks of spaceflight caused a decrease in succinate dehydrogenase activities of ventral horn and dorsal root ganglion neurons in rats. Significant effects on motor performance are also induced in both human (Kozlovskaya et al., 1981) and rats (Canu and Falempin, 1997), but these changes are reversible. However, it is not known how neuromuscular function respond to long-term gravitational unloading. Therefore, the current study was carried out to investigate the effects of 9 weeks of hindlimb suspension on the neuromuscular function and mass in hindlimb muscles in rats.     

Effects of nine weeks of unloading on neuromuscular activities in adult rats

Effects of 9-week hindlimb suspension and 8-week recovery on locomotor performance and electromyogram (EMG) activities of soleus (Sol), plantaris (Pl), lateral gastrocnemius (LG), and tibialis anterior (TA), were studied in adult rats. Hyperextension of knee and ankle joints, noted after nine weeks of suspension, did not recover during 8-week ambulation. Growth of Sol was fully inhibited by suspension and did not recover completely within 8 weeks of ambulation. The EMG levels in Sol, Pl, and LG 1 day after suspension were 52-76% less than the pre-suspension level (resting on the floor). These activities were recovered to or near the pre-suspension level around 1 week, but decreased again to 10-29% of controls from 7 to 9 weeks. The integrated EMG of TA was elevated during the first week of suspension but then gradually declined to control levels within four weeks. At the end of suspension, the Sol and Pl, not the LG, EMGs remained reduced and the TA EMG remained hyperactive. Co-activation of dorsi- and plantar- flexors occurred often during quadripedal walking following suspension. Such a phenomenon was not observed in the control rats. These phenomena were recovered within 1 week. It is suggested that the unloading-related alterations of neuromuscular activities and/or locomotion, but not the hyperextension of knee and ankle joints, in rats are reversible.     

Effects of Resveratrol on the Recovery of Muscle Mass Following Disuse in the Plantaris Muscle of Aged Rats

Aging is associated with poor skeletal muscle regenerative ability following extended periods of hospitalization and other forms of muscular disuse. Resveratrol (3,5,4’-trihydroxystilbene) is a natural phytoalexin which has been shown in skeletal muscle to improve oxidative stress levels in muscles of aged rats. As muscle disuse and reloading after disuse increases oxidative stress, we hypothesized that resveratrol supplementation would improve muscle regeneration after disuse. A total of thirty-six male Fisher 344 × Brown Norway rats (32 mo.) were treated with either a water vehicle or resveratrol via oral gavage. The animals received hindlimb suspension for 14 days. Thereafter, they were either sacrificed or allowed an additional 14 day period of cage ambulation during reloading. A total of six rats from the vehicle and the resveratrol treated groups were used for the hindlimb suspension and recovery protocols. Furthermore, two groups of 6 vehicle treated animals maintained normal ambulation throughout the experiment, and were used as control animals for the hindlimb suspension and reloading groups. The data show that resveratrol supplementation was unable to attenuate the decreases in plantaris muscle wet weight during hindlimb suspension but it improved muscle mass during reloading after hindlimb suspension. Although resveratrol did not prevent fiber atrophy during the period of disuse, it increased the fiber cross sectional area of type IIA and IIB fibers in response to reloading after hindlimb suspension. There was a modest enhancement of myogenic precursor cell proliferation in resveratrol-treated muscles after reloading, but this failed to reach statistical significance. The resveratrol-associated improvement in type II fiber size and muscle mass recovery after disuse may have been due to decreases in the abundance of pro-apoptotic proteins Bax, cleaved caspase 3 and cleaved caspase 9 in reloaded muscles. Resveratrol appears to have modest therapeutic benefits for improving muscle mass after disuse in aging.     

Rat cardiovascular responses to whole body suspension: head-down and non-head-down tilt.

The rat whole body suspension technique mimics responses seen during exposure to microgravity and was evaluated as a model for cardiovascular responses with two series of experiments. In one series, changes were monitored in chronically catheterized rats during 7 days of head-down tilt (HDT) or non-head-down tilt (N-HDT) and after several hours of recovery. Elevations of mean arterial (MAP), systolic, and diastolic pressures of approximately 20% (P < 0.05) in HDT rats began as early as day 1 and were maintained for the duration of suspension. Pulse pressures were relatively unaffected, but heart rates were elevated approximately 10%. During postsuspension (2-7 h), most cardiovascular parameters returned to presuspension levels. N-HDT rats exhibited elevations chiefly on days 3 and 7. In the second series, blood pressure was monitored in 1- and 3-day HDT and N-HDT rats to evaluate responses to rapid head-up tilt. MAP, systolic and diastolic pressures, and HR were elevated (P < 0.05) in HDT and N-HDT rats during head-up tilt after 1 day of suspension, while pulse pressures remained unchanged. HDT rats exhibited elevated pretilt MAP and failed to respond to rapid head-up tilt with further increase of MAP on day 3, indicating some degree of deconditioning. The whole body suspended rat may be useful as a model to better understand responses of rats exposed to microgravity.     

Role of afferent input in muscle atrophy.

It is well known that soleus muscle of rat atrophies following spaceflight or hindlimb suspension (Ohira et al., 1992). It is, further, reported that the electromyogram (EMG) of soleus muscle disappears immediately in response to unloading by exposure to actual micro-g environment (Kawano et al., 2002; Leterme and Falempin, 1998) and by hindlimb suspension of rats (Alford et al., 1987; Ohira et al., 2000). However, the EMG level is increased gradually to the control level following 7-10 days of continuous hindlimb suspension (Alford et al., 1987; Ohira, 2000), while muscle atrophy is progressing (Winiarski et al., 1987). We previously reported that reduction of the EMG level of rat soleus in response to actual micro-g environment, created by a parabolic flight of a jet airplane, was closely associated with a decrease of the afferent input recorded at the L5 segmental level of spinal cord (Kawano et al., 2002). However, it is still unclear how the EMG level of soleus muscle adapts to unloading condition. The current study was performed to investigate the responses of soleus EMG and both afferent and efferent neurogram at the L5 segmental level of spinal cord to acute (20 seconds) and chronic (14 days) unloading.     

Jump exercise during remobilization restores integrity of the trabecular architecture after tail suspension in young rats.

Three-dimensional trabecular architecture was investigated in the femora of tail-suspended young growing rats, and the effects of jump exercise during remobilization were examined. Five-week-old male Wistar rats (n = 35) were randomly assigned to five body weight-matched groups: tail-suspended group (SUS; n = 7); sedentary control group for SUS (S(CON); n = 7); spontaneous recovery group after tail suspension (S+R(CON), n = 7); jump exercise group after tail suspension (S+R(JUM); n = 7); and age-matched control group for S+R(CON) and S+R(JUM) without tail suspension and exercise (S(CON)+R(CON); n = 7). Rats in SUS and S(CON) were killed immediately after tail suspension for 14 days. The jump exercise protocol consisted of 10 jumps/day, 5 days/wk, and jump height was 40 cm. Bone mineral density (BMD) of the femur and three-dimensional trabecular bone architecture at the distal femoral metaphysis were measured. Tail suspension induced a 13.6% decrease in total femoral BMD (P < 0.001) and marked deterioration of trabecular architecture. After 5 wk of free remobilization, femoral BMD, calf muscle weight, and body weight returned to age-matched control levels, but trabeculae remained thinner and less connected. On the other hand, S+R(JUM) rats showed significant increases in trabecular thickness, number, and connectivity compared with S+R(CON) rats (62.8, 31.6, and 24.7%, respectively; P < 0.05), and these parameters of trabecular architecture returned to the levels of S(CON)+R(CON). These results indicate that suspension-induced trabecular deterioration persists after remobilization, but jump exercise during remobilization can restore the integrity of trabecular architecture and bone mass in the femur in young growing rats.     

Differential effects of jump versus running exercise on trabecular architecture during remobilization after suspension-induced osteopenia in growing rats

High-impact exercise is considered to be very beneficial for bones. We investigated the ability of jump exercise to restore bone mass and structure after the deterioration induced by tail suspension in growing rats and made comparisons with treadmill running exercise. Five-week-old male Wistar rats (n = 28) were randomly assigned to four body weight-matched groups: a spontaneous recovery group after tail suspension (n = 7), a jump exercise group after tail suspension (n = 7), a treadmill running group after tail suspension (n = 7), and age-matched controls without tail suspension or exercise (n = 7). Treadmill running was performed at 25 m/min, 1 h/day, 5 days/wk. The jump exercise protocol consisted of 10 jumps/day, 5 days/wk, with a jump height of 40 cm. Bone mineral density (BMD) of the total right femur was measured by dual-energy X-ray absorptiometry. Three-dimensional trabecular bone architecture at the distal femoral metaphysis was evaluated using microcomputed tomography. After 5 wk of free remobilization, right femoral BMD, right hindlimb muscle weight, and body weight returned to age-matched control levels, but trabeculae remained thinner and less connected. Although both jump and running exercises during the remobilization period increased trabecular bone mass, jump exercise increased trabecular thickness, whereas running exercise increased trabecular number. These results indicate that restoration of trabecular bone architecture induced by jump exercise during remobilization is predominantly attributable to increased trabecular thickness, whereas running adds trabecular bone mass through increasing trabecular number, and suggest that jumping and running exercises have different mechanisms of action on structural characteristics of trabecular bone.     

Effects of torbafylline on muscle atrophy: prevention and recovery.

The effects of torbafylline on the prevention of and the recovery from 5 weeks of hindlimb suspension induced atrophy were analyzed in rat soleus and extensor digitorum longus muscles. Muscle alterations were investigated by determining a suite of electrophysiological, histochemical, and muscle ultrastructural characteristics. Administration of torbafylline during the suspension period was ineffective in preventing any of the observed muscle atrophic changes. Application of torbafylline during the recovery period resulted in a faster recovery of some soleus muscle structural and functional properties. Mitochondrial volume densities and capillary to fiber ratios returned towards baseline values earlier in the recovery process with torbafylline. Furthermore, the drug significantly improved soleus muscle fatigue resistance 4 weeks after cessation of hindlimb suspension.     

Differential effects of post-natal development, animal strain and long term recovery on the restoration of neuromuscular function after neuromyotoxic injury in rat

We have analysed the effect of long term recovery, post-natal development and animal strain on the extent of restoration of neuromuscular function after neuromyotoxic injury in the rat (Rattus norvegicus). Muscle isometric contractile properties of soleus muscle in response to nerve stimulation were measured in situ in snake venom injured muscles and compared to contralateral uninjured muscles. We show here that neuromuscular function was not fully recovered until 24 weeks after injury in young adult (2-3 month old) Wistar rats. Moreover, the level of functional recovery 3 weeks after injury induced in juvenile rats (1 month old) was not globally different from that in younger adult, adult (10 month old) and older adult (24 month old) Wistar rats. Furthermore, the level of recovery of some contractile parameters differed between Wistar and Sprague-Dawley strains 3 weeks after injury. In conclusion, a very long time (>12 weeks) is required for full neuromuscular recovery following neuromyotoxic injury of young adult rats. Moreover, neuromuscular recovery during post-natal development is not markedly different from that during adult stage in the Wistar rat strain. Finally, some rat strain differences are observed in the recovery after injury of young adult rats.     

Effects of gravitational loading on rat soleus muscle fibers following hindlimb suspension.

Effects of 16 days of hindlimb suspension and 16 days of ambulation recovery at 1-G or 2-G environment on the characteristics of soleus muscle fibers were studied in male Wistar Hannover rats. The mean cross-sectional area and myonuclear number in isolated single fibers at the termination of suspension were approximately 30% and 25% of the controls, respectively. Satellite cells were distributed evenly throughout the fiber length in the control. However, the number of satellite cells distributed at the middle of the fiber was less in the unloaded rats immediately after the termination of suspension. Both the numbers of quiescent and mitotic active satellite cell per fiber were approximately 57% less immediately after the termination of suspension than controls. The number of satellite cells at the end of fibers was increased first during the early phase of reloading. Subsequently, the number at the middle was gradually increased. The myonuclear number per fiber was also less (approximately 25%) in the unloaded than the age-matched control at the termination of suspension, but was increased following the recovery. Although the mean in vivo sarcomere length of the soleus muscle was shortened in response to plantarflexion of ankle joint, the length at the certain ankle joint angle was increased after 16 days of suspension due to sarcomere remodeling. The length at the proximal and distal, rather than the middle, portion of the fiber was stretched in both reloaded and control rats in response to dorsiflexion of the ankle joint. But it was noted that the magnitude of stretch was greater in the unloaded rats. It is suggested that the fiber end is more stimulated rapidly than the middle portion by the load applied to the muscle during the ambulation recovery.     

Morphological and morphometric study on the effect of simulated microgravity on rat testis

The present study was undertaken to determine the effect of simulated microgravity on the testis of the rats and to evaluate the possibility of spermatogenesis failure in space environment. Fifty-four adult male albino rats were used in this study. They were divided equally into intact control, stress control and experimental groups. The rats of the intact control group (Group Ia) were kept without intervention. The rats of both the stress control and experimental groups were subjected to inguinal canal ligation and tail-suspension. In the stress control group (Group Ib) the hindlimbs were not elevated above the floor of the housing units whereas in the experimental groups the hindlimbs were elevated for one week (Group II) and six weeks (Group III), respectively. In a third experimental group (Group IV) the rats were hindlimb-suspended for six weeks followed by another six weeks without suspension to allow recovery. Prior to sacrifice, the animals were weighed and anesthetized, and the testes were excised and weighed. Testicular specimens were processed for histological, histochemical and morphometric studies. The results of the present study revealed that only after six weeks of hindlimb-suspension, the rats showed a significant decline in testicular weight compared with the control groups. Histologically, few abnormalities were observed in some seminiferous tubules in one-week hindlimb-suspended group. Spermatogenesis was significantly reduced by six-week of hindlimb-suspension marked by atrophy of the testes and loss of all germ cells, except a few spermatogonia. Spermatogenesis was partially restored in the recovery group. In all groups the appearance of Sertoli cells remained the same. Proliferation of Leydig cells was observed in the experimental groups. It is concluded that spermatogenesis is severely inhibited by six weeks of hindlimb-suspension and that it is partially restored following six weeks of recovery. This study provides further insights regarding the serious effects of long-term exposure to microgravity on the testes of mammals, including human beings.     

The effects of simulated weightlessness on bone biomechanical and biochemical properties in the maturing rat

Histomorphometric and biomechanical changes in bone resulting from hypogravity (simulated weightlessness) were examined in this study. Using a head-down hindlimb suspension model, three groups of six male rats underwent simulated weightlessness for periods of one, two and three weeks while a fourth recovery group was suspended for two weeks followed by two weeks of normal activity. Biomechanical data were collected during static and dynamic bending and torsion tests on intact femora. Histomorphometric values were determined from midshaft bone cross sections and material properties were obtained using ash and calcium assays. The experimental groups exhibited significantly lower geometric and material properties than the controls, resulting in structural hypotrophy; geometric and material changes contributed equally to the structural changes. Recovery following a return to normal activity was indicated, although full recovery may take longer than the weightlessness period. In the rat, altered maturation and reduced bone strength were the sequelae of weightlessness.     

Skeletal muscle response to spaceflight, whole body suspension, and recovery in rats

Comparisons of soleus and extensor digitorum longus (EDL) muscles from male Sprague-Dawley rats (350-400 g) after 7 days of weightlessness, 7 and 14 days of whole body suspension (WBS), and 7 days of recovery from WBS and from vivarium controls were made. Muscle mass loss of approximately 30% was observed in soleus after 7 and 14 days of WBS. Measurement of slow- and fast-twitch fibers showed significant alterations. Reductions in cross-sectional areas and increases in fiber densities in soleus after spaceflight and WBS were related to previous findings of muscle atrophy during unloading. Capillary density also showed a marked increase with unloading. Seven days of weightlessness were sufficient to effect a 20 and 15% loss in absolute muscle mass in soleus and EDL, respectively. However, the antigravity soleus was more responsive in terms of cross-sectional area reductions. After 7 days of recovery from WBS, with normal ambulatory loading, the parameters studied showed a reversal to control levels. Muscle plasticity, in terms of fiber and capillary responses, indicated differences in responses in the two types of muscles and further amplified that antigravity posture muscles are highly susceptible to unloading. Studies of recovery from spaceflight for both muscle metabolism and microvascular modifications are further justified.     

Muscle damage induced by running training during recovery from hindlimb suspension: the effect of dantrolene sodium

We examined the extent of morphological alterations and the myosin heavy chain (MHC) distribution in the rat soleus muscle after a 4-week period of spontaneous recovery or retraining after hindlimb suspension (HS). Moreover, we tested the hypothesis that dantrolene sodium, which affects the flux of calcium over the sarcoplasmic reticulum membrane, was able to attenuate muscle damage. Three groups of rats were submitted to 3 weeks of HS, followed by either 4 weeks of unrestricted cage activity (HC, n = 7), or running training for the same period and were compared to age-matched animals (C, n = 8). Trained rats were treated with either placebo or dantrolene sodium (HTP, HTD, n = 8 each, respectively). Four weeks after HS recovery, the percentage of myofibres with internal nuclei (%in) was determined by histological staining with hematoxylin and eosin. %in was affected by the individual rat (P < 0.001), and was higher in the mid-belly region of the muscle (P < 0.05). Muscle damage, as estimated by %in, was more extensive in trained rats (i.e. HTP and HTD) than in HC animals (23% and 12%, respectively). Moreover, dantrolene sodium tended to exert a protective effect on training-induced muscle injury. A 12% increase in type I MHC was observed in both HTP and HTD rats, in comparison with group C animals (P < 0.001). The relative proportion of type-I MHC was inversely correlated with %in (r = −0.65, P < 0.001). Running recovery led to an increased citrate synthase activity in comparison with that of C or HC rats. In conclusion, the present findings demonstrate that running recovery from HS increases the incidence of muscle damage, and that dantrolene sodium administration has only limited protective effects against exercise-induced muscle injury. Accepted: 29 April 1997     

Observations on arteriolar network structure in soleus of tail-suspended rats

Researchers studied the effects of weightlessness simulation on rat hindlimb muscle arterioles to determine changes in arteriole remodeling and if those changes are reversible. After four weeks of hindlimb suspension, arteriole diameter was examined at immediately after and at 1- and 5-weeks post suspension. Arteriole diameters were significantly reduced immediately after suspension with recovery of arcade and transverse II arterioles by 5 weeks. Transverse V arterioles were not fully recovered at 5 weeks post suspension.     

Intrastriatal Grafts of Fetal Mesencephalic Cell Suspensions in MPP+-Lesioned Rats: A Microdialysis Study In Vivo

The striatum of rats was lesioned by unilateral administration of MPP⁺. Two weeks later, a suspension of fetal mesencephalic cells (FMC), obtained from 14-day rat embryos, was injected into the lesioned striatum. Two weeks after grafting, the success of implantation and recovery of dopamine function were assessed by tyrosine hydroxylase immunocytochemistry (TH) and the measurement of striatal dopamine content. In addition, the extracellular concentrations of dopamine and dopamine metabolites were studied by microdialysis in vivo before and after perfusion of MPP⁺ to induce dopamine release from vesicular stores. TH+ cell bodies were seen in the lesioned grafted striata, indicating that fetal cells survived in these striata. In addition, there was a marked increase in TH-immunoreactivity in the neuronal fibers and terminals in the area surrounding the cell implant, suggesting a compensatory response of the host tissue which may involve fiber sprouting. Grafting induced a recovery in indices of dopamine function, including recovery in dopamine content, and basal and MPP⁺-induced dopamine release. Thus, grafts of FMC may provide a significant recovery of dopamine function in MPP⁺-lesioned striata.     

Changes of bone morphology in response to hindlimb suspension of rats.

Effects of chronic hindlimb suspension or exposure to 2-G from postnatal day 4 to month 3 followed by ambulation recovery on the floor on the morphology of hindlimb bones were investigated in rats. The dorsi-flexion of the ankle was inhibited in the suspended group and such phenomena were not recovered at all. The mean weight and length of femur, tibia, and fibula were less than the cage controls at the end of suspension, but gradually increased during recovery. However, they were still less than those of the age-matched controls even after 3-month recovery. External bending of shaft and rotation of distal end of tibia were observed in the suspended group and these phenomena were not recovered at all. These morphological changes caused the inhibition of dorsi-flexion of ankle joints. The electromyogram activities of ankle plantar-flexors, soleus, plantaris, lateral gastrocnemius, were inhibited and those of dorsi-flexor, tibialis anterior, were increased during suspension. Typical changes in bone morphology were not induced by exposure to 2-G. It was suggested that gravitational unloading during developing period causes irreversible inhibition of normal bone growth. It was also indicated that the suspension-related changes in bone morphology may be caused by abnormal mechanical stress due to the altered mobilization of hindlimb muscles.     

Quantitative assessment of degenerative changes in soleus muscle after hindlimb suspension and recovery

The aim of this study was to quantify the degenerative and regenerative changes in rat soleus muscle resulting from 3-week hindlimb suspension at 45° tilt (HS group, n = 8) and 4-week normal cage recovery (HS-R group, n = 7). Degenerative changes were quantified by microscope examination of muscle cross sections, and the myosin heavy chain (MHC) composition of soleus muscles was studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis. At the end of 3-week hindlimb suspension, histological signs of muscle degenerative changes were detected in soleus muscles. There was a significant variability in the percentage of fibres referred to as degenerating (%dg) in individual animals in the HS group [%dg = 8.41 (SEM 0.5)%, range 4.66%–14.08%]. Moreover, %dg varied significantly along the length of the soleus muscle. The percentage of fibres with internal nuclei was less than %dg in HS-soleus muscles [4.12 (SEM 0.3)%, range 1.24%–8.86%]. In 4-week recovery rats, the greater part of the fibres that were not referred to as normal, retained central nuclei [15.8 (SEM 2.2)%, range 6.2%–21.1%]. A significant increase in the slow isoform of MHC was recorded in the HS-R rats, compared to muscles from age-matched rats (P < 0.01). These results would suggest that a cycle of myofibre degeneration-regeneration occurred during HS and passive recovery, and that the increased accumulation of slow MHC observed in soleus muscles after recovery from HS could be related to the prevalence of newly formed fibres. Accepted: 14 October 1996     

Recovery of skeletal muscle mass after extensive injury: positive effects of increased contractile activity

The present study was designed to test the hypothesis that increasing physical activity by running exercise could favor the recovery of muscle mass after extensive injury and to determine the main molecular mechanisms involved. Left soleus muscles of female Wistar rats were degenerated by notexin injection before animals were assigned to either a sedentary group or an exercised group. Both regenerating and contralateral intact muscles from active and sedentary rats were removed 5, 7, 14, 21, 28 and 42 days after injury (n = 8 rats/group). Increasing contractile activity through running exercise during muscle regeneration ensured the full recovery of muscle mass and muscle cross-sectional area as soon as 21 days after injury, whereas muscle weight remained lower even 42 days postinjury in sedentary rats. Proliferator cell nuclear antigen and MyoD protein expression went on longer in active rats than in sedentary rats. Myogenin protein expression was higher in active animals than in sedentary animals 21 days postinjury. The Akt-mammalian target of rapamycin (mTOR) pathway was activated early during the regeneration process, with further increases of mTOR phosphorylation and its downstream effectors, eukaryotic initiation factor-4E-binding protein-1 and p70(s6k), in active rats compared with sedentary rats (days 7-14). The exercise-induced increase in mTOR phosphorylation, independently of Akt, was associated with decreased levels of phosphorylated AMP-activated protein kinase. Taken together, these results provided evidence that increasing contractile activity during muscle regeneration ensured early and full recovery of muscle mass and suggested that these beneficial effects may be due to a longer proliferative step of myogenic cells and activation of mTOR signaling, independently of Akt, during the maturation step of muscle regeneration.     

Moderately high folic acid supplementation exacerbates experimentally induced liver fibrosis in rats

Under certain clinical circumstances, folic acid can have undesirable effects. We investigated the following: (i) the effects of moderately high folic acid supplementation on the course of liver impairment in CCl(4)-treated rats and (ii) the influence of folic acid supplements on the hepatic recovery following the interruption of the CCl(4)-induced toxic injury. Four experimental groups of rats were used: CCl(4)-treated rats (0.5 ml of CCl(4) twice a week i.p.) fed standard chow for up to 12 weeks (Group A); treated rats fed chow supplemented with 25 mg/kg folic acid from weeks 6 to 12 (Group B); treated rats fed a standard diet but with CCl(4) discontinued after 6 weeks to allow for tissue recovery over 4 weeks (Group C); rats as Group C but fed a diet supplemented with 25 mg/kg folic acid from weeks 6 to 10 (Group D). Liver and blood samples were obtained for biochemical, histological, and gene expression analyses. Animals that received the supplement had a higher content of collagen, activated stellate cells, and apoptotic parenchymal cells in biopsy tissue at weeks 8 and 10 of treatment and more extensive alterations in serum albumin and bilirubin concentrations (Group B vs. Group A). In some of the time periods analyzed, alterations were observed in the expression of genes related to apoptosis (B-cell leukemia/lymphoma 2, inhibitor of apoptosis 2) and to fibrosis (procollagen I, matrix metalloproteinase 7). In the recovery period (Groups C and D), folic acid administration was associated with increased hepatic inflammation and apoptosis and with a decrease in the tissue inhibitor of metalloproteinase-3 expression following 1 week of recovery. We conclude that folic acid administration aggravates the development of fibrosis in CCl(4)-treated rats. Follow-up studies are needed to determine whether folic acid treatment would be contraindicated in patients with chronic liver diseases.