Effect of hypertension and peroxynitrite decomposition with FeTMPyP on CBF and stroke outcome

Abstract

We investigated the effect of peroxynitrite decomposition catalyst FeTMPyP treatment on perfusion deficit, vascular function and stroke outcome in Wistar (n = 26) and spontaneously hypertensive rats stroke-prone (SHRSP; n = 26) that underwent tMCAO for 2 h or Sham operation. Peri-infarct CBF was measured by hydrogen clearance in the absence or presence of FeTMPyP (10 mg/kg, i.v.) or vehicle 10 min before reperfusion. Myogenic tone of parenchymal arterioles (PAs) was measured as an indication of small vessel resistance (SVR). Baseline CBF was similar between Wistar and SHRSP (114 ± 12 vs. 132 ± 9 mL/100 g/min); however, MCAO caused greater perfusion deficit in SHRSP (24 ± 6 vs. 7 ± 1 mL/100 g/min; p < 0.05) and increased infarct volume by TTC (12 ± 6 vs. 32 ± 2%; p < 0.05). Reperfusion CBF was decreased from baseline in both SHRSP and Wistar (54 ± 16 and 46 ± 19 mL/100 g/min; p < 0.05), suggesting increased infarction in SHRSP was related to greater perfusion deficit. PAs from SHRSP had increased tone vs. Wistar that was enhanced after tMCAO. FeTMPyP treatment did not affect CBF during ischemia or reperfusion, or tone of PAs, but decreased the incidence of hemorrhage in SHRSP by 50%. Thus, increased tone in PAs from SHRSP could increase perfusion deficit during MCAO that was not alleviated by FeTMPyP.

Introduction

Hypertension is highly prevalent in the stroke population. A large-scale international clinical study of more than 17,000 patients showed that up to 82% of acute ischemic stroke patients were hypertensive (≥140/90 mm Hg).¹ The presence of hypertension not only increases the risk of stroke but also significantly worsens stroke outcome.^2–7 Elevated blood pressure in stroke patients is associated with greater death and disability after stroke.⁸ In addition, animals with genetic or renal hypertension have smaller ischemic penumbra and larger ischemic lesions than normotensive counterparts.^6,7

The underlying mechanism by which stroke outcome is worse during hypertension is likely multi-factorial. However, one of the most important consequences of hypertension is its effect on the cerebral vasculature. Increased tone and structurally smaller lumens of cerebral pial arteries and arterioles in response to hypertension is well documented to increase cerebrovascular resistance (CVR) and shift the autoregulatory curve to higher pressures.^9,10 Recently, other segments of the cerebral vasculature have been studied and found to be highly vasoconstricted during hypertension as well, including parenchymal arterioles (PAs) and pial collaterals (leptomeningeal anastomoses, LMAs).^11,12 Vasoconstriction of these vessels during hypertension could significantly increase perfusion deficit and limit penumbral flow during acute stroke. In addition, PAs from normotensive rats were shown to undergo vasoconstriction in response to early post-ischemic reperfusion^13,14 that could impair reperfusion CBF as well; however, whether this occurs during hypertension to worsen outcome is not known.

In the present study, we used SHRSP to investigate the effect of chronic hypertension on brain parenchymal CBF during ischemia and reperfusion to determine if hypertension was associated with greater perfusion deficit and incomplete reperfusion. We also sought to determine if changes in CBF during ischemia and reperfusion correlated with changes in the function and structure of PAs prior to and after post-ischemic reperfusion. In addition, we treated animals with peroxynitrite decomposition catalyst FeTMPyP prior to reperfusion because we previously showed that treatment of normotensive Wistar rats with FeTMPyP prevented constriction of PAs after ischemia and reperfusion.¹⁴ We hypothesized that treatment with FeTMPyP prior to reperfusion would improve reperfusion CBF and infarct in SHRSP, and be associated with decreased tone of PAs.

Materials and methods

Animals

Experiments were performed using male Wistar rats (Harlan, Indianapolis, IN, USA) and SHRSP (Charles River, Wilmington, MA, USA) that were 16–19 weeks old. All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Vermont and complied with the National Institutes of Health guidelines for care and use of laboratory animals. Rats were housed in the Animal Care Facility at the University of Vermont, an Association for Assessment and Accreditation of Laboratory Animal Care-accredited facility. Rats were maintained on a 12-h light/dark cycle and allowed food and water ad libitum. The reporting of this study was conducted in accordance with ARRIVE guidelines.

Model of transient focal ischemia

Transient proximal middle cerebral artery occlusion (tMCAO) was performed using the intravascular filament model, as previously described.¹³ Briefly, animals were anesthetized with isoflurane in oxygen (1.5–2%) and mechanically ventilated to maintain blood gases within normal physiologic ranges (Table 1). Two femoral artery catheters were inserted for monitoring blood pressure and obtaining blood gas samples. The MCA was occluded for 2 h of ischemia followed by filament removal to allow reperfusion for 30 min for isolated PA experiments or 2 h for stroke outcome and CBF experiments. Isoflurane anesthesia was used in all experiments except for those measuring CBF by hydrogen clearance. Laser Doppler flowmetry (Perimed Inc., Ardmore, PA, USA) was used to confirm MCAO and reperfusion. Animals were excluded if the drop in cerebral blood flow was < 60% from baseline. Sham control animals were exposed to isoflurane anesthesia for 2.5 h and received a midline neck incision, without filament insertion.

Table 1.

Body weights and arterial blood gases of all animals during Sham and MCAO surgeries.

		Arterial blood gases
	Weight (g)	pH	pCO2	pO2
Parenchymal flow & FeTMPyP treatment study
Wistar–Vehicle (n = 7)	360 ± 8	7.41 ± 0.01	38.8 ± 0.9	126.6 ± 6.6
Wistar–FeTMPyP (n = 7)	334 ± 12	7.39 ± 0.01	40.6 ± 1.3	125.6 ± 5.0
SHRSP–Vehicle (n = 7)	310 ± 6	7.35 ± 0.01	41.6 ± 1.9	118.2 ± 4.1
SHRSP–FeTMPyP (n = 6)	321 ± 6	7.34 ± 0.01	44.2 ± 1.4	120.0 ± 9.1
Isolated parenchymal arteriole study
Wistar–Sham (n = 6)	475 ± 56	7.44 ± 0.01	41.5 ± 1.4	103.7 ± 7.6
Wistar–MCAO (n = 5)	462 ± 45	7.44 ± 0.02	42.8 ± 2.0	102.8 ± 6.9
SHRSP–Sham (n = 6)	319 ± 10	7.46 ± 0.01	39.6 ± 1.2	107.6 ± 6.4
SHRSP–MCAO (n = 6)	340 ± 7	7.45 ± 0.02	39.3 ± 2.4	95.0 ± 8.8

Hydrogen clearance measurement of parenchymal CBF during MCAO

Parenchymal CBF was monitored during ischemia and reperfusion using hydrogen clearance. Animals (n = 8 in each group) were anesthetized with 2% isoflurane for instrumentation and tapered to chloral hydrate (300 mg/kg, i.v.).¹⁵ Animals were mechanically ventilated to maintain blood gases within normal physiologic ranges (Table 1). A burr hole was placed at the right temporal fossa carefully to not disturb the dura. A small diameter (50 µm) glass hydrogen sensor (Unisense, Aarhus, Denmark), which contained both the sensing anode and reference electrode together, was inserted 2 mm into the peri-infarct region of the MCA territory (Figure 1(a)). Hydrogen gas (4%) was inhaled until tissue saturation determined when hydrogen current reached a steady state and then ceased. Tissue desaturation of hydrogen was recorded by Multimeter (Unisense, Aarhus, Denmark) and the half-life of hydrogen calculated (Figure 1(b)).¹⁶ Local CBF was determined by first-order clearance rate of hydrogen gas locally in brain parenchyma. CBF by hydrogen clearance was measured at baseline and at several time points during 2 h of ischemia and 2 h of reperfusion.

Figure 1.

(a) Coronal section of a rat brain illustrating the placement of the hydrogen sensor for hydrogen clearance measurement of CBF. (b) Representative original tracing of tissue hydrogen saturation and clearance from which CBF was determined.

Treatment with peroxynitrite decomposition catalyst

To investigate the effect of peroxynitrite decomposition on reperfusion CBF (n = 8), PA tone (n = 6) and stroke outcome (n = 8), peroxynitrite decomposition catalyst FeTMPyP (10 mg/kg) or saline vehicle was infused intravenously via femoral catheter at 10 min prior to reperfusion.¹⁷

Determination of stroke outcomes

Following 2 h of reperfusion, animals were quickly decapitated under isoflurane anesthesia and the brain removed for measurement of infarct volume using 2,3,5-triphenyltetrazolium chloride (TTC) staining (n = 8 in each groups). Briefly, brains were removed and sliced to 2 mm coronal sections. Brain sections were incubated for 30 min at 37℃ in 2% TTC in phosphate buffered saline to stain for infarction, and subsequently fixed in 3.7% PBS-buffered formalin at 4℃ for 45 min for imaging using a digital scanner. The infarct area of each section was measured with ImageJ software (NIH, Bethesda, MD, USA) and corrected for edema by subtracting the contralateral from the ipsilateral hemisphere. Infarct volume was calculated by summing the edema-corrected infarct areas of the sections for each brain by investigators blinded to the groups.

We also observed and recorded the presence of hemorrhage in all animals. During the TTC procedures, the investigator observed the brain infarct area for presence of obvious pinkish red coloration after the brains were sliced to 2 mm coronal sections. The presence of obvious pink color within the infarct area, that was normally white, was identified as hemorrhage.

Preparation of isolated parenchymal arterioles and measurement of myogenic tone

Following 30 min of reperfusion, animals (n = 6 in each group) were quickly decapitated under isoflurane anesthesia and the brains removed and placed in cold, oxygenated physiologic saline solution (PSS). PAs, which branched perpendicularly off the MCA and penetrated into the brain tissue, were dissected from the M2 region of the ipsilateral MCA, mounted onto glass cannulas in an arteriograph chamber and secured with silk suture, as previously described.¹³ PAs were pressurized to 40 mm Hg and equilibrated for 1 h to allow spontaneous development of myogenic tone. After the equilibration period, myogenic reactivity was assessed with stepwise increases in intravascular pressure up to 100 mm Hg, and lumen diameter recorded once stable. At the conclusion of the experiment, PAs were superfused with diltiazem (10 µmol/L) in calcium-free PSS to obtain fully passive diameters.

Drugs and solutions

All isolated vessel experiments were performed using a bicarbonate-based Ringer’s PSS, the ionic composition of which was (mmol/L): NaCl 119.0, NaHCO₃ 24.0, KCl 4.7, KH₂PO₄ 1.18, MgSO₄ × 7H₂O 1.17, CaCl₂ 1.6, EDTA 0.026, and glucose 5.5. Zero calcium PSS was the same PSS composition without the addition of CaCl₂. PSS was made each week and stored without glucose at 4℃. Glucose was added to the PSS prior to each experiment. PSS was aerated with 5% CO₂, 10% O₂, and 85% N₂ to maintain pH. Diltiazem was purchased from MP Biomedicals (Santa Ana, CA, USA) and made up weekly to a 1 mmol/L stock. Peroxynitrite decomposition catalyst FeTMPyP (Cayman Chemicals, Ann Arbor, MI, USA) was dissolved in sterile lactated Ringer solution prior to use. NS309 was purchased from Sigma (St. Louis, MO, USA) and aliquots of 10 mmol/L stocks frozen at −20℃. TTC and formalin were purchased from Sigma.

Data calculations and statistical analysis

The number of animals used in each experiment was justified by statistical power calculation based on our previous similar studies.^13,17 Results are presented as mean ± SEM. Myogenic tone was calculated as a percent decrease in diameter from the fully relaxed diameter in calcium-free PSS with diltiazem by the equation: (1 − (ϕ_tone/ϕ_passive)) × 100%; where ϕ_tone is the inner diameter of the vessel with tone and ϕ_passive is the inner diameter of the vessel in calcium-free PSS with diltiazem. Parenchymal CBF was calculated by the rate of hydrogen clearance using the initial slope to estimate first-order clearance rate in which flow (mL/s) = 0.693/t_1/2 where t_1/2 is the time in seconds to reach half of the maximal tissue concentration of hydrogen, and then converted to mL/100 g tissue/min.¹⁶ Unpaired t-test or one-way analysis of variance (ANOVA) with a post hoc analysis for multiple comparisons was used to determine statistical differences between two groups or three or more groups, respectively. Two-way ANOVA was used to determine the effect of strain (Wistar vs. SHRSP) and intervention (MCAO vs. Sham), or strain and treatment (vehicle vs. FeTMPyP). Differences were considered significant at P < 0.05.

Results

CBF during tMCAO and stroke outcome in Wistar and SHRSP

Figure 2(a) shows CBF in SHRSP and Wistar rats measured using hydrogen clearance. One animal from each group was excluded due to technical difficulties during the procedure. Mean arterial pressure at the femoral artery was 133 ± 9 mmHg in SHRSP compared to 104 ± 9 mmHg in Wistar under anesthesia. Baseline CBF was similar between SHRSP and Wistar rats (Figure 2(a)). However, CBF during ischemia was significantly reduced in SHRSP compared to that of Wistar rats. In fact, CBF during ischemia was 24 ± 6 mL/100 g tissue/min in Wistar rats, a level considered to be able to sustain the penumbra.^18,19 In contrast, CBF during ischemia in SHRSP was 7 ± 1 mL/100 g tissue/min, which is considered core infarct flow.^18,19 All animals reperfused when the filament was removed; however, reperfusion CBF was significantly reduced compared to baseline in both SHRSP and Wistar to a similar level. Thus, incomplete reperfusion occurred in both normotensive and hypertensive animals, regardless of the degree of ischemia. In addition, infarct volume in SHRSP was significantly increased compared to Wistar (Figure 2(b)).

Figure 2.

Effect of hypertension on CBF and infarct volume. (a) Cerebral blood flow at baseline and during ischemia and reperfusion in Wistar and SHRSP using hydrogen clearance. Flow measurements were taken 30 and 60 min after MCAO and 15, 30, 60 and 90 min after reperfusion. SHRSP had greater perfusion deficit than Wistar during MCAO and both groups had incomplete reperfusion; (b) brain infarct volume measured using TTC after 2 h of ischemia and 2 h of reperfusion in the same animals as in (a); and (c) the relationship between reperfusion CBF at 30 min and brain infarct volume in Wistar and SHRSP. There was a positive relationship in both groups such that greater reperfusion CBF was related to better stroke outcome. Results are mean ± SEM. *p < 0.05 vs. Wistar; ^p < 0.05 vs. baseline, using t-test.

To investigate the relationship between reperfusion CBF and infarction, linear regression analysis and calculation of the Pearson correlation coefficient between early reperfusion CBF at 30 min and infarct volume was performed. We found a positive correlation for Wistar (coefficient at 0.472) and SHRSP (coefficient at 0.703) (Figure 2(c)), suggesting early reperfusion was beneficial to stroke outcome. The parallel, but upward shifted regression line for SHRSP was due to their larger infarct volumes, but the correlation was similar. Thus, decreased infarct volume was related to better early post-ischemic reperfusion CBF in both Wistar and SHRSP.

Myogenic reactivity and tone of PAs after ischemia and reperfusion in Wistar and SHRSP

Previous studies demonstrated increased tone of PAs and enhanced SVR in response to post-ischemic reperfusion that could contribute to impaired reperfusion.^20–22 However, it is unknown if a similar effect occurs in hypertension. We therefore determined if PAs had increased vasoconstriction after MCAO in hypertensive animals. Figure 3(a) shows that diameters of PAs from SHRSP-Sham and SHRSP-MCAO were smaller compared to those from Wistar at all pressures studied. One animal from SHRSP-Sham group was excluded due to technical issue during Sham operation. In addition, PAs from both groups of SHRSPs had increased tone compared to Wistar (Figure 3(b)). Two-way ANOVA revealed that ischemia and reperfusion significantly increased myogenic tone of PAs at 40 mm Hg in both SHRSP and Wistar groups. Thus, both hypertension and MCAO significantly increased myogenic tone of PAs. Interestingly, PAs from SHRSP were not structurally smaller than those from Wistar rats (Figure 3(c)), suggesting increased myogenic tone was the main contributor to the smaller diameters of PAs in SHRSP.

Figure 3.

Effect of hypertension and ischemia and reperfusion on function and structure of PAs. (a) Inner diameter of PAs in response to increasing intravascular pressure. PAs were taken from Wistar and SHRSP after 2 h of ischemia and 30 min of reperfusion or sham control. Inner diameters of PAs from both groups of SHRSP were smaller than PAs from Wistar rats; (b) Percent myogenic tone of PAs at 40 mm Hg in the same groups as in (a). Both MCAO and hypertension caused an increase in tone; and (c) passive inner diameters of PAs at pressures from 5–100 mm Hg in Wistar and SHRSP that underwent tMCAO. There was no difference in passive inner diameters suggesting the smaller diameters from SHRSP were due to increased tone and not structural remodeling. Results are mean ± SEM. *p < 0.05 vs. Wistar; ^p < 0.05 vs. Sham using two-way ANOVA.

CBF during tMCAO and stroke outcome in Wistar and SHRSP: effect of FeTMPyP

Our finding in isolated PA experiments suggested an increase in SVR prior to and during ischemia and reperfusion in chronic hypertension that could contribute to increased perfusion deficit and infarct volume. We previously showed that treatment of PAs from normotensive rats with FeTMPyP after ischemia and reperfusion prevented enhanced vasoconstriction, suggesting peroxynitrite production caused vasoconstriction.¹⁴ We therefore sought to determine if FeTMPyP treatment could alleviate the vasoconstriction and improve reperfusion CBF in hypertensive rats. Figure 4(a) shows CBF at 60 min of ischemia and at 30 min of reperfusion in untreated or FeTMPyP-treated rats. One animal from Wistar and two animals from SHRSP treated with FeTMPyP were excluded due to technical difficulties during MCAO or hydrogen clearance procedure. FeTMPyP treatment did not affect CBF during ischemia or reperfusion in either Wistar or SHRSP. FeTMPyP also had no significant effect on infarct volume in SHRSP that was increased compared to that of Wistar rats (Figure 4(b)). Similarly, brain edema formation in SHRSP was also significantly increased compared to that of Wistar rats that was not affected by FeTMPyP treatment (Figure 4(c)).

Figure 4.

Effect of peroxynitrite decomposition catalyst FeTMPyP treatment on CBF and stroke outcome. (a) CBF at 60 min of ischemia and at 30 min of reperfusion measured using hydrogen clearance in Wistar and SHRSP that had FeTMPyP or vehicle treatment. CBF during ischemia was significantly decreased in SHRSP vs. Wistar rats and FeTMPyP treatment did not improve perfusion deficit in either group. Reperfusion CBF at 30 min was similar between Wistar and SHRSP and not affected by FeTMPyP; (b) infarct volume in the same groups as in (a) measured by TTC. FeTMPyP did not affect infarct that was increased in SHRSP; and (c) swelling (edema) measured from the same animals as (b) using TTC-stained sections. FeTMPyP did not affect brain edema in either group that was greater in SHRSP. Results are mean ± SEM. *p < 0.05 vs. Wistar using two-way ANOVA.

Hemorrhagic transformation after ischemia and reperfusion

We noted considerable hemorrhagic transformation after MCAO in SHRSP animals only. We therefore determined if FeTMPyP treatment could prevent this secondary brain injury. Figure 5(a) shows a representative image of a whole brain and a coronal section with hemorrhagic transformation obtained from a SHRSP after ischemia and reperfusion. Hemorrhage was only observed in SHRSP with and without treatment with FeTMPyP and not in Wistar rats (Figure 5(b)). FeTMPyP treatment of SHRSP rats decreased the incidence of hemorrhagic transformation from 57% to 33%.

Figure 5.

Hemorrhagic transformation in Wistar and SHRSP after ischemia and reperfusion. (a) Representative images of a whole brain and brain coronal section from SHRSP with hemorrhage within the infarct area. (b) The incidence of hemorrhage in vehicle- or FeTMPyP-treated Wistar (Wistar + Fe) and SHRSP (SHRSP + Fe). Hemorrhage occurred only in SHRSP animals. Treatment with FeTMPyP decreased the incidence of hemorrhage in that group. (c) CBF measured using hydrogen clearance in SHRSP with and without hemorrhage. There was no difference in the ischemic blood flow between those animals that had hemorrhage and those that did not; (d) reperfusion CBF measured using hydrogen clearance in SHRSP with and without hemorrhage. Animals that had hemorrhage had significantly lower reperfusion CBF; (e) percent infarct volume in SHRSP with and without hemorrhage. Animals with hemorrhage had a nonsignificant increase in infarct volume; (f) swelling (edema) in SHRSP with or without hemorrhage. There was no difference in the amount of edema in those that had hemorrhage vs. those that did not. Results are mean ± SEM. *p < 0.05 vs. no hemorrhage using t-test.

To better understand what factors contribute to hemorrhage in SHRSP, we compared CBF levels during ischemia and reperfusion in animals with and without hemorrhage. Ischemic CBF was similar in SHRSP with or without hemorrhage (Figure 5(c)), and low in both groups. However, SHRSP that underwent hemorrhagic transformation had decreased reperfusion CBF, demonstrating the beneficial effect of early reperfusion (Figure 5(d)). In fact, animals with hemorrhage had reperfusion CBF that was only around 20 mL/100 g tissue/minute, whereas animals without hemorrhage had reperfusion CBF that was >50 mL/100 g tissue/min.

We also determined if hemorrhagic transformation was associated with worse stroke outcome including infarct volume and edema formation. Infarct volume was non-significantly increased in the hemorrhage group (p = 0.08) (Figure 5(e)); however, edema (swelling) was similar regardless of hemorrhage (Figure 5(f)).

Discussion

The main findings of this study were that SHRSP had increased perfusion deficit during ischemia, incomplete reperfusion CBF, and enhanced infarction compared to normotensive Wistar rats. Moreover, we have shown for the first time that tone was increased, which could increase SVR and contribute to these hemodynamic alterations during stroke in hypertensive rats. SHRSP also had hemorrhagic transformation that was not present in Wistar rats. Treatment with FeTMPyP did not alleviate any of the CBF alterations in SHRSP during MCAO but did decrease the incidence of hemorrhagic transformation, suggesting peroxynitrite may have a role in BBB breakdown in SHRSP during postischemic reperfusion.

Previous studies have also shown increased perfusion deficit and small penumbra in hypertensive animals. Both genetic (SHR) and renal hypertension were shown to have large ischemic cores and smaller ischemic penumbra than normotensive rats using perfusion–diffusion mismatch on MRI as early as 30 min of ischemia.⁶ Using a similar imaging approach, SHRSP were shown to have increased ischemic damage and perfusion deficit that increased over time compared to normotensive WKY rats.⁷ In this previous study,⁷ the small penumbra remained static in SHRSP while perfusion deficit increased. The results of the current study are in agreement with these previous studies in that SHRSP had increased perfusion deficit and infarction compared to Wistar (Figure 2(a)). In fact, CBF in the peri-infarct region just 30 min after ischemia was already at levels considered to be core infarction in SHRSP, but were at levels that would be considered penumbral flow in Wistar.

Hypertension is associated with increased vasoconstriction of cerebral arteries and arterioles that could increase perfusion deficit and decrease the area of salvageable tissue. In particular, LMAs are thought to sustain flow to the penumbra by promoting retrograde perfusion from the anterior cerebral artery territory to the MCA territory, i.e. from the unobstructed to obstructed vascular territories.^23,24 We recently demonstrated that hypertension caused LMAs to have considerable (>50%) myogenic tone, but were not structurally smaller.¹¹ This was in contrast to LMAs from normotensive rats that had little tone (<10%), a state that would be conducive to retrograde flow. We speculate that the large ischemic core and small penumbra in SHRSP may be partly due to hyperconstricted LMAs. It is also possible that increased tone of PAs in SHRSP contributed to increased perfusion deficit and infarction in those animals. PAs were not found to be structurally smaller in SHRSP, an effect that if present would be expected to decrease vasodilatory reserve during ischemia and increase perfusion deficit. Thus, increased tone of LMAs and PAs could both be contributing to the large ischemic core and limited penumbral tissue in SHRSP.

This study also found that, similar to our previous study, PAs had increased tone after ischemia and reperfusion compared to sham controls that could contribute to incomplete reperfusion through increased SVR. Incomplete reperfusion is common and a major issue in treating acute stroke because although vessel recanalization is strongly associated with better stroke outcome and reduced mortality,²⁵ recanalization does not always result in complete reperfusion of downstream tissues.^25–27 Clinical studies showed that up to only about half of acute ischemic stroke patients had complete reperfusion after recanalization.^28–30 Although several mechanisms have been proposed to explain the “no reflow” phenomenon, there is considerable evidence that SVR is increased at early time points of reperfusion when other cell processes proposed to be involved in no reflow, such as immune cell activation, would not be a large contributor.^17–19,31 A previous histological study found that pre-capillary arterioles in the brain parenchyma were smaller in the areas of no-reflow than the adjacent reflow areas.²⁰ In addition, experimental studies showed that intra-cortical vascular resistance was increased at post-ischemic reperfusion, suggesting constriction of arterioles in the brain parenchyma that can impede flow during reperfusion.^21,22 Since PAs are high-resistance vessels in the brain that directly connect the pial network to the capillaries, their vasoconstriction during reperfusion could have a major impact on reducing downstream flow.^32,33 However, treatment with FeTMPyP did not alleviate the incomplete reperfusion, despite our previous study showing that it could prevent vasoconstriction of PAs after MCAO in vitro.¹⁴ FeTMPyP treatment also did not alleviate the increase in perfusion deficit or improve infarction in either Wistar or SHRSP. The reason for the lack of effect of FeTMPyP is not clear but may be related to dosing that was too low or that peroxynitrite is not a major player in incomplete reperfusion.

Although FeTMPyP treatment did not improve reperfusion or stroke outcome in SHRSP, it did have a modest effect on hemorrhagic transformation. Blood–brain barrier disruption is another major injury associated with chronic hypertension.³⁴ SHRSP have increased susceptibility to blood–brain barrier damage even at young age (∼20 weeks old).^31,35 Blood–brain barrier damage in SHRSP appears to be exacerbated following ischemia and reperfusion that explains the presence of hemorrhagic transformation in these animals only.³⁶ The presence of hemorrhagic transformation was associated with lower reperfusion CBF, suggesting that early but adequate reperfusion CBF prevented hemorrhagic transformation. This agrees with previous studies in animals showing that early post-ischemic reperfusion (within 3 h of ischemia) is protective of the blood–brain barrier while delayed reperfusion (>3 h) may cause further damage.^37–40 The current study is distinct in that all animals had early reperfusion (after 2 h of ischemia) but not all animals reperfused to the same degree. It was only animals that did not reperfuse well that had hemorrhagic transformation. This finding highlights another detrimental effect of incomplete reperfusion that of hemorrhagic transformation under conditions that promote it such as hypertension. It is interesting that in addition to early, adequate reperfusion, treatment with FeTMPyP reduced the incidence of hemorrhagic transformation by 50%. FeTMPyP has been shown to inhibit matrix metalloproteinases 2 and 9 activation that may underlie the protective effect of FeTMPyP on the blood–brain barrier in SHRSP.⁴¹

In conclusion, chronic hypertension was associated with greater brain injury compared to normotensive rats that appeared to be due to a combination of increased perfusion deficit and reduced reperfusion CBF. In fact, low reperfusion CBF appeared to cause hemorrhagic transformation in SHRSP. Hypertension-induced constriction of PAs and LMAs and consequent increased vascular resistance may be involved in worse perfusion deficit and small penumbra during MCAO in SHRSP. Peroxynitrite decomposition catalyst FeTMPyP did not effectively improve CBF or infarction during acute ischemic stroke in either normotensive or hypertensive animals, but might be beneficial to hemorrhagic transformation in chronic hypertension.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Heart, Lung, and Blood Institute grant (grant no. P01 HL095488), the National Institute of Neurological Disorders and Stroke Grant (grant no. R01 NS093289), and by the Totman Medical Research Trust.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Authors’ contributions

MJC designed the study, analyzed data, and wrote the manuscript; SLC performed experiments, analyzed data, compiled figures, and wrote parts of the manuscript; JGS performed experiments, analyzed data, compiled figures, and wrote parts of the manuscript.