Abstract
Bedridden patients develop atrophied muscles, their daily activities greatly reduced, and some display a depressive mood. Patients who are able to receive physical rehabilitation sometimes show surprising clinical improvements, including reduced depression and attenuation of other stress-related behaviors. Regenerative medicine has advanced two major stem cell-based therapies for CNS disorders, namely, transplantation of exogenous stem cells and amplification of endogenous neurogenesis. The latter strategy embraces a natural way of reinnervating the damaged brain and correcting the neurological impairments. In this study, we discussed how immobilization-induced disuse atrophy, using the hindlimb suspension model, affects neurogenesis in rats. The overarching hypothesis is that immobilization suppresses neurogenesis by reducing the circulating growth or trophic factors, such as vascular endothelial growth factor or brain-derived neurotrophic factor. That immobilization alters neurogenesis and stem cell differentiation in the CNS requires characterization of the stem cell microenvironment by examining the trophic and growth factors, as well as stress-related proteins that have been implicated in exercise-induced neurogenesis. Although accumulating evidence has revealed the contribution of “increased ” exercise on neurogenesis, the reverse paradigm involving “lack of exercise, ” which mimics pathological states (e.g., stroke patients are often immobile), remains underexplored. This novel paradigm will enable us to examine the effects on neurogenesis by a nonpermissive stem cell microenvironment likely produced by lack of exercise. BrdU labeling of proliferative cells, biochemical assays of serum, cerebrospinal fluid and brain levels of trophic factors, growth factors, and stress-related proteins are proposed as indices of neurogenesis, while quantitative measurements of spontaneous movements will reveal psychomotor components of immobilization. Studies designed to reveal how in vivo stimulation, or lack thereof, alters the stem cell microenvironment are needed to begin to develop treatment strategies for enhancing neurogenesis in bedridden patients.
Introduction
Our overarching hypothesis is that lack of exercise alters the neurogenic niche in the brain, thereby altering the stem cell microenvironment. Until recently, the nonregenerative capability of the adult damaged brain was an accepted scientific dogma. However, accumulating evidence indicates that neurons and astrocytes can be generated from isolated cells of the adult mammalian central nervous system (CNS) (46). Several laboratory studies have examined stem cell therapy for treating various diseases in the CNS, including stroke, traumatic brain injury, and neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. Stem cell therapy, however, remains as an experimental treatment. Many people continue to suffer from these diseases, and the number of bedridden patients is increasing. Bedridden patients develop atrophied muscles, their daily activities are greatly reduced, and some patients present with a depressive mood. It is also recognized in the clinic that patients who are able to receive good rehabilitation sometimes show surprising clinical improvements, including reduced depression and attenuation of other stress-related behaviors. However, very little information is available about the effects of disuse atrophy upon intrinsic potencies of brain, including neurogenesis.
Regenerative medicine has emerged as a new scientific field advancing stem cell therapy for treating brain disorders, with emphasis on either transplanting exogenous stem cells or amplifying endogenous stem cells via neurogenesis (22, 41-43, 57-64). The proposed research project focuses on the latter one, which incorporates a natural way of reinnervating the damaged brain and correcting the neurological impairments via physical rehabilitation. We discuss how lack of exercise-induced disuse atrophy, using the hindlimb suspension (HS) model, affects neurogenesis in adult rats. The interaction between lack of exercise and neurogenesis remains to be fully characterized; however, the reversed model demonstrating that increased exercise enhances neurogenesis has been previously examined (54). In particular, exercise has been shown to induce neurogenesis in the dentate gyrus (DG) of the hippocampus (17, 21, 54). In parallel, some diseased states, like cerebral ischemia, have also been demonstrated to increase neurogenesis (49). In consideration of enhanced neurogenesis induced by exercise, the overlying concept of our thesis is that lack of exercise suppresses neurogenesis possibly by reducing circulating factors, such as vascular endothelial growth factor (VEGF) or brain-derived neurotrophic factor (BDNF). Laboratory studies are warranted to elucidate the biological mechanisms underlying neurogenesis, and such in-depth understanding should provide insights into development of strategies designed to augment neurogenesis in patients with lack of mobility.
The Hindlimb Suspension Model
The lack-of-exercise model using HS was initially proposed for studying spaceflight-associated phenomena (37), based largely on the initial data demonstrating inhibition of bone formation during spaceflight (38). Thereafter, different modifications of the model have been introduced (20); in addition to bone formation studies (3), investigations on muscle (7) and vascular network of the hindlimbs (20) have been performed. To date, most studies have focused on peripheral effects of HS (39, 40, 44). There are only a few studies using the HS model to reveal changes in the CNS, with most focusing on depression (12, 14). In 2005, Dupont and colleagues demonstrated that nerve growth factor (NGF) and BDNF mRNA as well as NGF protein were upregulated in the somatosensory cortex of animals exposed to HS (16). These alterations in neurotrophic factor levels support the notion that “exercise ” (17, 54), or lack thereof, regulates neurotrophic factor expression. Although there exist no investigations of the HS model in CNS disorders, there are reports of forelimb disuse and overuse models in stroke and Parkinson's disease models (8, 9, 10, 11). In stroke rats, forced disuse (using one-sleeved casts), but not overuse, of the affected forelimb in the early phase of stroke recovery worsens the functional outcome (6). In rats with somatosensory cortical lesions, however, forced overuse of the impaired forelimb during the early stages of recovery exacerbated the functional outcome (26). Dr. Schallert's group, who reported these incongruent results, explained that the differential effects of forelimb disuse and overuse may be due to the site-specific anatomical changes (e.g., subcortical in stroke vs. cortical in the somatosensory cortical lesion model) produced by such injury paradigm (47). Accordingly, subcortical rather than cortical injury appears to benefit from forced use of the affected forelimb. Of note, the forced use of the impaired forelimb immediately after unilateral 6-hydroxydopamine (6-OHDA) lesions in rats attenuated behavioral deficits (51); similarly, such forced forelimb regimen prior to 6-OHDA lesion increased glial cell-derived neurotrophic factor (GDNF) levels and protected against behavioral deficits (13). The observed amelioration of behavioral impairments in both stroke and Parkinson's disease animal models corresponded with sparing of ischemia-and lesion-induced neuroanatomical and neurochemical deficits (2, 8-11, 28, 29). These studies clearly indicate the utility of facilitating or withholding physical therapy, via forced forelimb overuse or disuse, in examining alterations in the CNS, including diseased states. Here we provide the rationale for characterizing CNS effects of regulating physical therapy using the HS model.
Neurogenesis and Growth Factors
In Dr. Gage and colleagues' pioneering studies on exercised-induced neurogenesis (53, 54), they showed that free access to running wheel for 3 h during the nocturnal period (active phase of rats) significantly increased 5-bromo-2-deoxyuridine (BrdU)-labeled newly formed cells in the subgranular zone (25). Voluntary exercise also reduced threshold for long-term potentiation (LTP) in the DG with subsequent enhanced LTP, suggesting enhanced memory function by exercise (18, 53). The exercise-induced neurogenesis coincided with increased levels of VEGF (17, 49) and BDNF (5, 18, 31, 36), suggesting the participation of these growth factors in exercise and neurogenesis. These findings indicate that neurotrophic factors are closely associated with and directly modulate the microenvironments of known neurogenic sites, such as the subventricular zone (SVZ) and DG. In addition, because these growth factors are diffusible molecules, nonneurogenic sites may also be affected by this exercise-induced neurogenesis. Indeed, a recent study (4) revealed that exercise altered neuronal activity in the posterior hypothalamic area (PHA), in that the in vitro and in vivo spontaneous firing rates of PHA neurons from exercised rats were significantly lower than those of nonexercised rats. To this end, exercise-induced neurogenesis has been recognized in neurogenic sites and potentially may stimulate neurogenesis-like neuronal activity in other nonneurogenic brain areas. Positive correlations between exercise and neurotrophic factor levels or neurogenesis suggest that manipulating the level of exercise would directly alter the level of neurotrophic factors and neurogenesis. Because a diminishing access to exercise appears to generally accompany the aging process and the progression of diseased CNS states, a fertile ground to examine the effects of exercise on neurotrophic factors and neurogenesis will be to utilize a lack-of-exercise paradigm. While it is established that absence of physical therapy leads to a variety of health problems (e.g., osteoporosis, obesity, cardiovascular diseases) (23, 35, 45, 55), the roles of neurotrophic factors and neurogenesis have yet to be examined in this paradigm. In order to begin to understand and develop therapeutic targets for correcting physical inactivity-mediated behavioral deficits, it will be important to identify critical growth factors, at least those present in the neurogenic sites, affected by lack of exercise. Of note, environmental restriction, with physical inactivity as a major component, triggers motor and cognitive functions and also exacerbates these behavioral deficits in diseased states (32, 56). By focusing on one or two critical parameters accompanying physical inactivity, one can approximate clinical/rehabilitation conditions that would be effective in either preventing the early onset of brain degeneration or facilitating the recovery from brain injury.
Neurogenesis and Stress
The principal parameters for characterizing the effects of lack of exercise include neurotrophic factors and stress proteins, which represent the two ends of a continuum. The hypothesis is that lack of exercise decreases neurotrophic factors while increasing stress proteins and should lead to reduced neurogenesis. Indeed, lack of exercise achieved by HS is accompanied by chronic stress (14). Chronic stress and depression increased glucocorticoids and decreased serotonin (19, 30, 50). Aging also increased glucocorticoids and decreased insulin-like growth factor-l (IGF-1). Both aging and chronic stress were shown to suppress neurogenesis, suggesting the important role of IGF-1 in neurogenesis (1). Along the same line, BDNF and serotonin in models of depression or chronic stress have been implicated in neuronal plasticity (27, 33, 34, 48). Thus, evaluating chronic stress, which likely accompanies the HS model, will provide insights into the role of stress in neurogenesis.
Neurological Effects of Deficient Exercise
Bed-restricted patients who receive adequate physical therapy are able to display clinical enhancement. A large amount of research suggests that exercise promotes endogenous neurogenesis and can possibly defend against CNS disorders. We previously investigated how lack of exercise affects neurogenesis in rats by using a standard HS model for a 2-week-long period. This process is comprised of raising the rat's tails, which raises their hindlimbs, and delivering the weight to the front of the rat. Also, exercise and recovery time was investigated in rats that received normal caging after HS. BrdU (50 mg/kg, IP), a chemical used to label proliferative cells, was injected every 8 h during the last 4 days of each model. Results from immunohistochemistry insisted that HS seriously decreased the amount of BrdU/doublecortin (Dcx) double-positive cells in both the SVZ and the DG zone of the brain. While exercise and recovery time remarkably enhanced atrophy of the soleus muscle, they did not lessen the HS-induced decrement in the BrdU/Dcx-positive cells. A different group of rats was exposed to an identical HS model, and an enzyme-linked immunosorbent assay (ELISA) of neurotrophic factors was executed on brain tissue that was harvested at the end of the HS periods. In addition, ELISA assays of neurotrophic factors were performed on plasma samples from all of the animals. These results suggested that HS reduced both the amount of BDNFs in the hippocampus and the VEGF plasma levels. It was learned in this study that lack of exercise decreases neurogenesis because of a shortage of neurotrophic factors. Using the HS model in addition to CNS disease models, the function of exercise in neurogenesis and neurotropic factors will further be clarified.
Rehabilitation and Neurogenesis
Through many research studies and observations, it has been well noted for years that the lack of physical therapy can lead to a multitude of health problems (e.g., osteoporosis, obesity, cardiovascular diseases) (23, 35, 45). The roles of neurotrophic factors and neurogenesis have yet to be examined in this scenario. Restrictions placed on the recovering individual's environment, with lack of physical activity being the major component, spawn abnormal motor and cognitive functions under healthy nonpathological conditions. If the recovering individual is diseased, then the lack of physical activity worsens the behavioral deficits (32, 56). Furthermore, although the usefulness of increased physical activity has been recognized in the clinic, such a therapeutic regimen has not been confirmed and sometimes was counterintuitive in diseased animal models. For example, 18 days of forced treadmill exercise shows no improvements to motor and memory functions involved with the upregulation of BDNF mRNA in CA1 and CA3, but not DG (24). Similarly, placing a non-impaired limb in a cast to increase the use of an impaired limb during rehabilitation after an injury to the sensori-motor cortex worsens the coordinated movement of both limbs (47). On the contrary, equally compelling evidence establishes that exercise improves the neuronal damage and motor functions in rodent neurologic disorder paradigms (e.g., stroke) (15, 52). The rehabilitation method with its duration, complexity, and frequency combined with the severity of the diseased condition of the patient and animal cases might alter the data. This alteration can lead to inconsistent results in future studies. The current recovery period of 2 or 4 weeks and exercise post-HS for 2 weeks constricted the mass of the soleus muscle toward standard levels. However, abolishing the HS-suppressed neurogenesis in the SVZ and DG was not enhanced by the recovery time and exercise, thus suggesting further optimization of rehabilitation techniques. When considering therapeutic candidates for optimizing physical inactivity-mediated behavioral deficits, our study provides valuable insights on the potential role of neurotrophic factors. In particular, BDNF and VEGF appear intimately associated with neurogenesis.
Conclusions
We have identified a novel paradigm—the HS model—for examining the role of withholding exercise in neurogenesis in the adult brain. Our rationale for focusing on neurotrophic factors and stress proteins in elucidating the effects of lack of exercise on neurogenesis is based on the existing paradigm of “increased exercise ” or “enriched environment, ” as well as on established indices for the HS model when used in peripheral injury (bone and muscle). In addition to the fact that this will be the first time to use HS model as a CNS model (i.e., neurogenesis), the novel scientific advance in this proposed study is our desire to provide a closer approximation of the aging brain and diseased brain states, which are both characterized by physical inactivity (Fig. 1). Accordingly, the lack of exercise paradigm will generate new information about neurogenesis that otherwise may be not well represented in the increased exercise or enriched environment model. In summary, preclinical studies are needed to assess alterations in neurogenesis in models of immobilized rats. Moreover, biomarkers of stem cell alterations need to be developed, including growth factors and stress-related proteins, underlying the anticipated lack of exercise-induced changes in neurogenesis.
Normal neurogenesis is recognized in the balanced condition of humoral factors. Adequate exercise might increase neurotrophic/growth factors and decrease stress-related hormones, resulting in enhanced neurogenesis. In contrast, the lack of exercise might decrease neurotrophic/growth factors and increase stress-related hormones, resulting in reduced neurogenesis.
Footnotes
Acknowledgments
Dr. Cesar V. Borlongan is funded by the National Institutes of Health (Grant 1R01NS071956-01A1), the Department of Defense (Grant W81XWH-11-1-0634), and the VA Merit Review.