Effects of Caffeine on Intervertebral Disc Cell Viability in a Whole Organ Culture Model.

STUDY DESIGN: Controlled laboratory study. OBJECTIVE: To investigate the impact of exposure to physiologically relevant caffeine concentrations on intervertebral disc (IVD) cell viability and extracellular matrix composition (ECM) in a whole organ culture model as potential contributing mechanisms in development and progression of IVD disorders in humans. Primary outcome measures were IVD viable cell density (VCD) and ECM composition. METHODS: A total of 190 IVD whole organ explants from tails of 16 skeletally mature rats-consisting of cranial body half, endplate, IVD, endplate, and caudal body half-were harvested. IVD explants were randomly assigned to 1 of 2 groups: uninjured (n = 90) or injured (20G needle disc puncture/aspiration method, n = 100). Explants from each group were randomly assigned to 1 of 3 treatment groups: low caffeine (LCAF: 5 mg/L), moderate caffeine (MCAF: 10 mg/L), and high caffeine (HCAF: 15 mg/L) concentrations. RESULTS: Cell viability was significantly higher in the low-caffeine group compared with the high-caffeine group at day 7 (P = .037) and in the low-caffeine group compared with the medium- and high-caffeine groups at day 21 (P ≤ .004). Analysis of ECM showed that all uninjured and control groups had significantly higher (P < .05) glycosaminoglycan concentrations compared with all injured groups. Furthermore, we observed a temporal, downward trend in proteoglycan to collagen ratio for the caffeine groups. CONCLUSIONS: Caffeine intake may be a risk factor for IVD degeneration, especially in conjunction with disc injury. Mechanisms for caffeine associated disc degeneration may involve cell and ECM, and further studies should elucidate mechanistic pathways and potential benefits for caffeine restriction.

Intralingual Administration of AAVrh10-miRSOD1 Improves Respiratory But Not Swallowing Function in a Superoxide Dismutase-1 Mouse Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by degeneration of motor neurons and muscles, and death is usually a result of impaired respiratory function due to loss of motor neurons that control upper airway muscles and/or the diaphragm. Currently, no cure for ALS exists and treatments to date do not significantly improve respiratory or swallowing function. One cause of ALS is a mutation in the superoxide dismutase-1 (SOD1) gene; thus, reducing expression of the mutated gene may slow the progression of the disease. Our group has been studying the SOD1G93A transgenic mouse model of ALS that develops progressive respiratory deficits and dysphagia. We hypothesize that solely treating the tongue in SOD1 mice will preserve respiratory and swallowing function, and it will prolong survival. At 6 weeks of age, 11 SOD1G93A mice (both sexes) received a single intralingual injection of gene therapy (AAVrh10-miRSOD1). Another 29 mice (both sexes) were divided into two control groups: (1) 12 SOD1G93A mice that received a single intralingual vehicle injection (saline); and (2) 17 non-transgenic littermates. Starting at 13 weeks of age, plethysmography (respiratory parameters) at baseline and in response to hypoxia (11% O2) + hypercapnia (7% CO2) were recorded and videofluoroscopic swallow study testing were performed twice monthly until end-stage disease. Minute ventilation during hypoxia + hypercapnia and mean inspiratory flow at baseline were significantly reduced (p < 0.05) in vehicle-injected, but not AAVrh10-miRSOD1-injected SOD1G93A mice as compared with wild-type mice. In contrast, swallowing function was unchanged by AAVrh10-miRSOD1 treatment (p > 0.05). AAVrh10-miRSOD1 injections also significantly extended survival in females by ∼1 week. In conclusion, this study indicates that intralingual AAVrh10-miRSOD1 treatment preserved respiratory (but not swallowing) function potentially via increasing upper airway patency, and it is worthy of further exploration as a possible therapy to preserve respiratory capacity in ALS patients.

5-HT2A/B receptor expression in the phrenic motor nucleus in a rat model of ALS (SOD1G93A).

Despite respiratory motor neuron death, ventilation is preserved in SOD1G93A rats. Compensatory respiratory plasticity may counterbalance the loss of these neurons. Phrenic long-term facilitation (pLTF; a form of respiratory plasticity) in naïve rats is 5-HT2 and NADPH oxidase-dependent. Furthermore, 5-HT2A, not 5-HT2B, receptor-induced phrenic motor facilitation is NADPH oxidase-independent in naïve rats. pLTF is NADPH oxidase-dependent in pre-symptomatic, but not end-stage, SOD1G93A rats. Here, we hypothesized that in the putative phrenic motor nucleus (PMN) of SOD1G93A rats vs. wild-type littermates: 1) pre-symptomatic rats would have greater 5-HT2B receptor expression that decreases at end-stage; and 2) 5-HT2A receptor expression would increase from pre-symptomatic to end-stage. Putative PMN 5-HT2A receptor expression was reduced when comparing across (but not within) pre-symptomatic vs. end-stage groups (p < 0.05). In contrast, putative PMN 5-HT2B receptor expression was increased when comparing across pre-symptomatic vs. end-stage groups, and within end-stage groups (p < 0.05). These data suggest a potential role for 5-HT2 receptors in pLTF and breathing in SOD1G93A rats.

Effects of ERß and ERα on OVX-induced changes in adiposity and insulin resistance.

Loss of ovarian hormones leads to increased adiposity and insulin resistance (IR), increasing the risk for cardiovascular and metabolic diseases. The purpose of this study was to investigate whether the molecular mechanism behind the adverse systemic and adipose tissue-specific metabolic effects of ovariectomy requires loss of signaling through estrogen receptor alpha (ERα) or estrogen receptor ß (ERß). We examined ovariectomized (OVX) and ovary-intactwild-type (WT), ERα-null (αKO), and ERß-null (ßKO) female mice (age ~49 weeks; n = 7-12/group). All mice were fed a phytoestrogen-free diet (<15 mg/kg) and either remained ovary-intact (INT) or were OVX and followed for 12 weeks. Body composition, energy expenditure, glucose tolerance, and adipose tissue gene and protein expression were analyzed. INT αKO were ~25% fatter with reduced energy expenditure compared to age-matched INT WT controls and ßKO mice (all P < 0.001). Following OVX, αKO mice did not increase adiposity or experience a further increase in IR, unlike WT and ßKO, suggesting that loss of signaling through ERα mediates OVX-induced metabolic dysfunction. In fact, OVX in αKO mice (i.e., signaling through ERß in the absence of ERα) resulted in reduced adiposity, adipocyte size, and IR (P < 0.05 for all). ßKO mice responded adversely to OVX in terms of increased adiposity and development of IR. Together, these findings challenge the paradigm that ERα mediates metabolic protection over ERß in all settings. These findings lead us to suggest that, following ovarian hormone loss, ERß may mediate protective metabolic benefits.