In this study, we found that density of TH (+) neurons and fibers in primary VM cultures were dose dependently reduced at 2 days after 6-hydroxydopamine treatment. 6-OHDA also induced DNA fragmentation as demonstrated in TUNEL labeling. These data are in agreement with our previous studies and suggest that administration of 6-OHDA causes activation of apoptotic pathways that lead to the death of VM dopaminergic neurons. We found that early and post treatment with 9cRA reduced 6-OHDA -mediated injury in VM cells. 6-OHDA -mediated reduction of TH cell density was significantly antagonized by 9cRA, suggesting that 9cRA is neuroprotective against 6-OHDA -mediated neurodegeneration in dopaminergic neurons in vitro.
Previous studies have demonstrated that RA exerts anti-apoptotic and antioxidant activity in neuronal and kidney cells. RA reduced staurosporine-induced oxidative stress and apoptosis by preventing the decrease in the levels of Cu-,Zn-superoxide dismutase (SOD-1) and Mn-superoxide dismutase (SOD-2) in primary hippocampal cultures  and by facilitating NGF-induced protection in chick embryonic neurons . RA suppressed hydrogen peroxide –induced apoptotic nuclear condensation and membrane blebbing in rat glomeruli mesangial cells. Our recent studies also indicated that 9cRA reduced the density of TUNEL labeling, a marker for apoptosis/necrosis in ischemic cortex after middle cerebral artery occlusion in rats . In this study, we demonstrated that treatment with 9cRA reduced 6-OHDA-mediated DNA fragmentation in culture. Taken together, our data suggest that 9cRA inhibits apoptosis and/or necrosis induced by 6-OHDA.
Using an established rodent model of PD, we also demonstrated the protective effect of 9cRA in vivo. Administration of amphetamine analogs causes ipislateral rotation in unilaterally 6-OHDA -lesioned rats due to differential increase of dopaminergic activity on the intact side. There is a correlation between amphetamine–induced ipislateral rotation and the depletion of dopamine in the nigra . In this study, 9cRA was given after rotation experiment on day 6 post lesioning. We separated animals to 2 groups to balance rotational behavior before treatment; the difference in lesioning prior to treatment was thus minimal between two groups. We demonstrated that post-treatment with 9cRA, compared to vehicle, significantly attenuated rotation. Our data support that early and post-treatment with 9cRA reduced behavioral imbalance in hemiparkinsonian rats.
The protective effect of 9cRA is further supported by in vivo electrochemical data at 2 months after lesioning. We used high speed chronoamperometry to examine the time course of KCl -evoked DA release and clearance in striatum. The dose of KCl applied locally was between 7–14 × 10-11 mole (70 mM × 204.0 ± 9.1 nl) per site. The dose, concentration and volume of the KCl solution have been previously reported to induce depolarization and release of dopamine at dopaminergic nerve terminals in vivo[26, 32]. We found that local administration of KCl induced DA release equally in the non-lesioned side striatum in 9cRA or vehicle –treated rats. In the lesioned side striatum, KCl-evoked DA release and the rate of DA clearance were greatly diminished in animals receiving vehicle treatment. Treatment with 9cRA did not induce a full recovery but significantly increased KCl-evoked DA release and DA clearance in the lesioned side striatum. These electrochemical data support that post-treatment with 9cRA protects against 6-OHDA –mediated deficiency in DA function in striatum.
Using unbiased stereology, we demonstrated that post-treatment with 9cRA significantly reduced the loss of TH cells in nigra. In contrast to a 95% reduction in TH cells in the lesion side nigra in animals with vehicle treatment, 70% TH (+) neurons were lost in the lesioned animals with 9cRA treatment. Similarly, there was about 99% reduction in KCl-evoked DA release in striatum in vehicle -treated animals. The reduction in DA release was only 66% after 9cRA treatment. Taken together, our data suggest that 9cRA has a protective effect against 6-OHDA injury in nigrostriatal dopaminergic neurons. It has been reported that the symptoms of the PD appear when 70-80% dopamine is depleted in patients [33, 34]. In this study, we demonstrated that early treatment with 9cRA can reduce the death of DA neurons to 70%. It is thus possible that post-treatment with 9cRA can be beneficial to PD patients by slowing down its progressive neurodegeneration.
We previously demonstrated that 9cRA can selectively induce BMP7 mRNA expression in the brain. In this study, we demonstrated that 9cRA reduced 6-OHDA -mediated program cell death in culture. BMP7 has anti-apoptotic properties. BMPs reduced caspase-3 activation and DNA fragmentation in the ischemic brain [35, 36] and attenuated dopaminergic neurotoxin -mediated apoptosis and cell death in nigrostriatal DA neurons. Interestingly, the reduction of TUNEL labeling by 9cRA in stroke brain was antagonized by the BMP antagonist noggin. Taken together, these data suggest that the protective effects of 9cRA may be indirectly mediated through a BMP signaling mechanism.
RA analogs, such as 9cRA and atRA, are agonists for RXR and RAR receptors. Compared to atRA, 9cRA is a more selective agonist for RXR. Although both atRA and 9cRA are neural protective against oxygen-glucose deprivation in hippocampal neurons , a differential sensitivity of these two ligands have been seen in other brain regions. Animals pretreated with 9cRA had lesser cortical infarction than those treated with atRA after middle cerebral artery occlusion . Using RTPCR, we also found that 9cRA is more potent than atRA in inducing BMP7 (unpublished observation) and midkine  expression in primary cortical cultures. We demonstrated here that 9cRA is more potent than atRA, at 50 nM, against 6-OHDA –mediated neurodegeneration in TH neuronal culture. Taken together, these differential responses of 9cRA suggest that neuroprotection induced by 9cRA involves activation of RXR.
Although 9cRA has a high affinity for RXR, it is not detectable in adult brain tissue and, thus, may not be a candidate endogenous ligand for this receptor. Studies have indicated that the polyunsaturated fatty acids, such as linolenic and docosahexaenoic acid, activate RXR [10, 38]. These polyunsaturated fatty acids have also been reported to reduce neurodegeneration [39, 40]. Future experiments are required to investigate the mechanism of protection induced by these polyunsaturated fatty acids and their effects on RXR in animal model of PD.
There are several limitations to deliver drug to brain days after onset of 6-OHDA lesioning. Drugs given systemically may not easily cross the blood brain barrier (BBB) and can be degraded through first pass metabolism. Intracerebral delivery is not feasible for repeated drug administration and may require chronic cannulation. Previous reports have indicated that small molecules can by-pass the BBB and reach brain parenchyma non-invasively through an intranasal delivery. In this study, 9cRA was given initially through i.c.v. and then repeatedly through an intra-nasal route. We found that intranasal administration of 9cRA increased brain 9cRA level to 3 ng/g or 10 nM at 1 hour after delivery as detected by LC-MS/MS analysis (Additional file 1:Figure S1). No 9cRA was seen after vehicle treatment. Previous studies have shown that 9cRA can activate RXR receptor at this concentration .
In a preliminary study, we treated 6-OHDA –lesioned rats with intranasal 9cRA without i.c.v injection from day 7 to day 14. We did not find significant behavioral improvement after intranasal 9cRA treatment and days 20 and 30, suggesting that a loading dose of 9cRA, given i.c.v. on day 7 is required for this protective reaction.