The current study confirms and extends our previous work in rats in which injection of the DA neurotoxin 6-hydroxydopamine into the striatum or medial forebrain bundle produced similar patchy areas of leakage of FITC-LA and horseradish peroxidase as well as increases in β3 integrin expression (a marker of angiogenesis) in the SN and striatum (Carvey et al., 2005
). In the present study, the DA neurotoxin (MPTP) was injected systemically so that the barrier dysfunction seen could not be a consequence of stereotaxic brain surgery. In addition, two different genotypes of mice exhibited barrier dysfunction using MPTP suggesting that the previous effects observed in rats were neither species nor toxin specific. Finally, the current study clearly demonstrated the validity of the FITC-LA injection procedure as a marker for barrier integrity, because areas devoid of a BBB exhibited leakage, regardless of treatment history, and detailed evaluation of several non-dopaminergic brain areas revealed complete vascular perfusion without evidence of leakage demonstrating specificity while ruling out a perfusion-based epiphenomenon. These data strongly argue that DA neurotoxins induce BBB dysfunction in well-established animal models of PD.
At this time, we do not know if the areas of leakage were static (i.e., an area developed a leak and remained leaky) or dynamic (i.e. an area developed a leak and repaired itself such that after several days, leakage would no longer be detected). Given that activated microglia were associated with areas of leakage and microglia are known to migrate (Cho et al., 2006
;Carbonell et al., 2005
), it is more likely that the pattern of leaks is dynamic. A dynamic pattern of leakage was further supported by the fact that the areas of leakage were different in every animal. This suggests that a “window” of increased leakage developed in an area and could subsequently close. In addition, at the present time we do not know if DA neurotoxins produce only “patchy” areas of severe dysfunction as indicated here with FITC-LA leakage (and as we observed in our previous study (Carvey et al., 2005
)), or if subtle dysfunction exists throughout the SN and striatum, but have not yet been identified. The large molecular weight of FITC-LA may only identify areas of significant leakage and if markers of smaller molecular weight were used, a much less punctated pattern of leakage might have been observed. Regardless, it is becoming increasingly clear that the BBB dysfunction, although it may be punctate and dynamic in nature, is not temporary. We previously showed that FITC-LA leakage was still present 10 and 34 days following 6-OHDA treatment (Carvey et al., 2005
). Whether or not barrier dysfunction continues for months after toxin exposure remains to be established. Regardless, these results argue that the BBB is somewhat long-lived and common to two different animal models of PD.
The MPTP-induced barrier dysfunction appeared to be a consequence of neuroinflammation independent of DA neuron loss. Although MPTP produced neuroinflammation as evidenced by increased numbers of activated microglia and increases in TNF-α and IL-1β in both genotypes, the TNF-α KO animals exhibited significant DA neuron losses whereas the animals treated with minocycline did not. Both TNF-α KO and minocycline treatment dramatically attenuated the FITC-LA leakage in which a clear-cut dissociation between DA neuron loss and barrier dysfunction was shown. However, we must consider the differential effects of MPTP on the BBB that was observed could simply reflect strain differences used in the two different studies. In addition, there is the possibility that TNF-α KO and minocycline treatment could have had differential effects on the expression of receptors on the endothelial cells or other soluble factors produced by MPTP exposure that prevented BBB dysfunction independent of DA neuron death. While not inherently apparent from the present results other factors originating from the dieing or compromised DA neurons could also be involved in the dysfunction of the BBB. Regardless, although biogenic amine containing neurons are often seen in close proximity to endothelial cells that also express noradrenergic and serotonergic receptors, and norepinephrine containing cell of the locus coeruleus have been shown to regulate barrier function (Kobayashi et al., 1985
;Raichle et al., 1975
;Wakayama et al., 2002
), there was no evidence of dopaminergic control of barrier leakage in the SN and striatum in the current study, after three days. We have previously shown that 6OHDA produces BBB dysfunction after 10 and 34 days (Carvey et al., 2005
), therefore there is a possibility that neuroinflammation can initiate a first phase of BBB dysfunction (i.e. 3 days) that is further perpetuated by the ensuing DA neuron loss (between 3 and 10 days). Detailed time course studies would be needed to address this possibility. Regardless neuroinflammatory events by themselves appear to lead to barrier dysfunction in the absence of DA neuron loss. Whether or not the magnitude of inflammation is correlated with the degree of BBB dysfunction cannot be determined with the current study design. Future studies should be performed to ascertain the levels of inflammation produced and correlate these with the degrees of BBB dysfunction. Regardless, the current results do not negate the probability that the DA neuron degeneration led to the inflammation, but rather, that DA neuron loss by itself did not lead to the barrier dysfunction. Moreover, since MPTP is widely known to primarily affect catecholaminergic systems in general and the nigro-striatal pathway in particular, it was not surprising to observe here as well as in our previous study that the significant BBB dysfunction was confined to the SN and striatum.
TNF-α by itself activates microglia (John et al., 2003
) and our results show that TNF-α KO inhibits microglia activation. Minocycline is a semisynthetic tetracycline derivative that exerts anti-inflammatory effects and inhibits microglial activation by decreasing IL-1 β and nitric oxide release (Du et al., 2001
;Tikka and Koistinaho, 2001
). Moreover, our data showed that minocycline also decreased the TNF-α release. Activated microglia release numerous neuroinflammatory substances that can potentially disrupt barrier function including prostanoids, proteases, nitric oxide (NO), superoxide, and the proinflammatory cytokines TNFα and IL-1β (Dringen, 2005
;Lynch et al., 2004
;Tsao et al., 2001
;Wong et al., 2004
;Yenari et al., 2006
). Although any of these factors could have contributed to the barrier breakdown in the current study, the MPTP-induced increase in TNF-α and IL-1β in conjunction with FITC-LA leakage and the attenuation in these increases in the TNF-α KO and minocycline treated animals argues for their potential involvement in the barrier disruption. TNF-α and IL-1β are known to decrease electrical resistance and increase permeability in BBB in vitro
models (Didier et al., 2003
;Miller et al., 2005
). TNF-α causes a redistribution of cadherin and junctional adhesion molecule (JAM) leading to a rearrangement of microfilaments, and a down-regulation of occludin expression increasing BBB permeability (Ozaki et al., 1999
;Petrache et al., 2003
;Kniesel and Wolburg, 2000
;Mankertz et al., 2000
). IL-1 β also increases BBB permeability by decreasing expression of occludin and zonula occludens-1 proteins leading to apparent redistribution of the adherens junction protein vinculin (Bolton et al., 1998
). IL-1β may also induce cyclooxygenase-2 (COX-2) synthesis and activate NFκB in brain endothelial cells, which can be blocked by specific inhibitors of NFκB activation (Kortekaas et al., 2005
;Laflamme et al., 1999
;Nadjar et al., 2005
). However, although the current evidence suggests the involvement of TNF-α and IL-1β, we cannot, at this time, rule out other inflammogens associated with microglial activation.
Several studies have argued against or have not found BBB dysfunction in PD. Haussermann et al., (2001)
found no changes in blood cerebral spinal fluid (CSF) barrier function by examining CSF/serum ratios and oligoclonal bands in PD patients. If the BBB dysfunction is not universal, but rather punctate as our data suggests, major changes such as these would not be detected. Similarly, Kurkowska-Jastrzebska et al., (1999)
argued that the DA neurotoxin MPTP does not disrupt BBB function. Yet, although they showed that IgG was restricted to the inside of the blood vessels, they reported that mononuclear cells infiltrated the SN and striatum, which would suggest BBB dysfunction. O’callaghan et al., (1990)
used a single small dose of MPTP (12.5mg/kg, s.c.) that did not produce DA neuron loss and reported no dysfunction of the BBB. Furthermore, Canudas et al., (2000)
into the left SN of the rat that produced only a small lesion, yet assessed BBB integrity in the striatum with a crude index of BBB integrity (albumin staining) that could have easily been missed because of the small size of the SN injury. It is also important to note that the animal studies using MPTP or its metabolite MPP+ just described, were not designed to assess BBB integrity, but rather focused on other effects of MPTP. Moreover, they used global indices of BBB integrity, which could have readily missed the punctuate leakage that seems to characterize the DA neurotoxin exposed BBB. In contrast, the study by Faucheux et al., (1999)
showed an increase in vascular density in the SN, but not the VTA of PD patients and Barcia et al., (2004)
discussed evidence of microangiogenesis in the PD brain which is often associated with barrier dysfunction. Kortekaas et al., (2005)
C]-verapamil imaging, and demonstrated an 18% increase in brain uptake in the mesencephalon of PD patients relative to aged controls. This increase may have reflected alterations in P-gp function or simply increased leakage into brain. Notably, Barcia et al., (2004)
also found an increase in the number of blood vessels indicative of microangiogenesis that follows barrier damage in close proximity to degenerating DA neurons in non-human primates. Moreover, the increase in vessels was highly correlated with increases in vascular endothelial growth factor (VEGF) probably caused by neo-microangiogenesis that similarly accompanies barrier brea kdown following an inflammatory event. Thus, this emerging literature supports barrier dysfunction in patients with PD and animal models of this disorder.
There are numerous implications to PD if areas of active inflammation lead to focal areas of barrier dysfunction in patients. Areas of leakage in focal neuroinflammatory loci would afford the opportunity to target deliver anti-inflammatory agents that normally do not cross the BBB or deliver a variety of therapeutics to those areas using nanoparticles. Notwithstanding the effect of these agents on areas of the brain not protected by a BBB, this strategy would target areas ostensibly undergoing active DA neurodegeneration potentially slowing disease progression. On the other hand, BBB dysfunction could allow inhomogeneous entry of antiparkinsonian drugs into the SN and striatum that could contribute to dyskinesias due to dopaminergic “hotspots” that have been implicated in this DA agonist induced side effect (Bankiewicz et al., 2006
). Alternatively, levodopa decarboxylase inhibitors including carbidopa and benserazide, which normally do not cross the BBB, may do so in these areas of leakage. This would inhibit conversion of levodopa to DA in these areas creating hot spots of increased DA activity in intact areas. In addition, focal BBB dysfunction would increase entry of elements of the peripheral vasculature into the SN and striatum that could similarly contribute to disease progression. Moreover, environmental toxins that may not cross the BBB readily would concentrate in areas of BBB dysfunction. Taken together, these results suggest that detailed imaging assessments of BBB integrity should be performed in patients with PD to determine the role, if any, the compromised BBB integrity plays in disease progression and side effects.