We used hypercapnia as an experimental tool to examine the regulation of cerebrovascular reactivity in the context of HIV-induced neuroinflammation. Our initial experiments used a relatively extended (5 minute) exposure to moderate hypercapnia, consistent with previous studies in other mouse and rat models of cerebrovascular reactivity (6% CO2 stimulus with 9-minute evaluation of CBF)
[45–47]. These studies demonstrated a significant loss of normal vascular responsiveness to hypercapnia in Tat-exposed mice, relative to controls (Figure
1). However, we noted that there was considerable acidemia in the animals at the end of this CO2 administration period (Table
1). Respiratory acidosis of this kind
 can stimulate systemic hemodynamic change through β-adrenergic signaling
[16, 17]. To avoid this, we therefore explored the administration of a 30-second CO2 challenge. This shorter hypercapnic exposure recapitulated our initial long exposure findings (compare Figures
1A with 1C), while minimizing the potential confounder of moderate respiratory acidosis (Table
We proceeded to use this hypercapnic challenge model to examine the regulation of CBF in the context of two experimental animal models for HIV-induced neuroinflammation. In both models, mice were exposed to HIV-1 Tat (either by direct intracranial injection or through enforced transgene expression within the CNS), in order to induce a neuroinflammatory state mimicking that found in persons with HIV-associated neurocognitive disorders. In both models, we detected significant reductions in cerebrovascular reactivity to brief hypercapnia, suggesting an underlying cerebrovascular pathology that was induced by exposure to HIV-1 Tat. Mice exposed acutely to intracranial Tat showed a more profound depression in the cerebrovascular response to hypercapnia compared to Tat-tg mice. This difference might be explained by a robust inflammatory response to acutely injected Tat, compared to a more slowly developing, chronic inflammation produced in the Tat-tg mouse during three weeks of DOX-induced Tat expression. Since HIV-associated neuroinflammation is also a chronic disease, all remaining experiments were conducted in the Tat-tg model.
Tat-tg mice showed a 50% attenuation of the normal increase in CBF, following exposure to hypercapnia. This suggests that the dysregulation of CBF in persons living with HIV-1 may be a direct result of virally-induced neurovascular inflammation, and not secondary to peripheral inflammation or peripheral vascular disease associated with HIV-1 infection. Additionally, these findings suggest that decreased resting CBF in persons living with HIV
 might be explained not only as a result of reduced neuronal demand (due to neuronal damage), but also as the consequence of an HIV-1 induced vascular dysfunction. Clinically, the failure to regulate CBF in the face of changing metabolic demand, such as increased CO2 levels, may in turn lead to transient episodes of cerebral ischemia, further perpetuating neuronal damage and inflammation.
In addition to examining regional cerebral blood flow using laser Doppler flowmetry, we also conducted experiments to look at the response of individual vessels to brief hypercapnia. To do this, we conducted in vivo two-photon imaging of cerebral blood vessels, using a thin-skull window (intact cranium). This allowed preservation of normal intracranial pressure, CSF dynamics and hemodynamics. We found that Tat-induced neuroinflammation reduced the overall magnitude of CO2-induced vasodilation, with an especially profound effect on larger vessels (>25 μm diameter) (Figure
Cerebrovascular responses to hypercapnia are believed to occur through an NO-cGMP pathway between perivascular neurons and smooth muscle cells
[19–23]. This pathway is highly susceptible to dysregulation by reactive oxygen species produced during inflammation
[48, 49]. Moreover, HIV-1 Tat has also been shown to significantly decrease endothelial NOS (eNOS) expression in porcine coronary arteries
. We therefore conducted experiments to examine the role of the NO-cGMP axis in contributing to the diminished vascular response to brief hypercapnia in mice with HIV-1 associated neuroinflammation.
First, we tested whether increasing the supply of NO might correct the vascular response to CO2. NO production by NOS is dependent on its cofactor tetrahydrobiopterin (BH4)
[26, 27]. When BH4 levels are inadequate, the reduction of O2 by NOS is no longer coupled to L-arginine oxidation, resulting in generation of superoxide rather than NO. To test whether this uncoupling of NOS was responsible for impaired CVR in our model, we supplemented Tat-tg and WT mice with BH4 and then exposed them to brief hypercapnia. BH4 had no effect on the cerebrovascular responses to brief hypercapnia, which remained attenuated in the Tat-tg mice. In contrast, when Tat-tg mice were treated with a PDE5 inhibitor (gisadenafil), expected to prevent degradation of cGMP in arterial smooth muscle cells
[24, 25], their cerebrovascular response to brief hypercapnia was normalized. This indicates that CO2 chemosensing was not impaired in the Tat-tg mice, and suggests that HIV-associated neuroinflammation may cause cerebrovascular pathology, through effects on cGMP metabolism and possibly PDE5. Interestingly, while gisadenafil produced significant recovery of the normal CBF response to brief hypercapnia in both small and large diameter vessels (Figure
5H, I, respectively), it showed selectivity for smaller vessels (<25 μm). One possible explanation is that the dilatation of the larger vessels is more reliant on NO
[51, 52], which may not have been adequately replenished with BH4 supplementation. An alternative explanation is that PDE5 expression may be limited to the smaller vessels, whereas other PDE family members exist within the larger arterioles. Future studies will need to address the large vessel responsiveness to different PDE inhibitors. More importantly, however, inhibition of PDE5 was sufficient to restore the normal increase in CVR. This suggests that responses in the smaller vasculature may be of greater importance, compared to larger arterioles (>25 μm), in overall control of CBF following brief hypercapnic stimulation. Additionally, it is possible that already available PDE5 inhibitors could be used to limit further neuroinflammation initiated by transient episodes of tissue ischemia, due to decreased CVR to CO2 or other CBF regulators signaling through the NO-cGMP pathway.
Overall, this study reports three major findings. First, HIV-associated neuroinflammation (induced by either acute or chronic exposure to HIV-1 Tat) produced a cerebrovascular pathology, that was uncovered upon brief exposure to moderate hypercapnia. Second, this cerebrovascular pathology may selectively affect larger vessels (>25 μm) and finally, inhibition of PDE5 by gisadenafil besylate restored the normal cerebrovascular response to transient hypercapnia in mice with underlying HIV-associated neuroinflammation by fully restoring the dilatory capacity of the smaller pial arterioles. These findings may have important implications for persons living with HIV-1 especially because impaired vasoreactivity to hypercapnia has been associated with increased risk of ischemic stroke, elevated overall cardiovascular risk and cognitive impairment