cerebrovascular structure and function after ... - Wiley Online Library

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J Physiol 594.6 (2016) pp 1677–1688

Rigid and remodelled: cerebrovascular structure and function after experimental high-thoracic spinal cord transection A. A. Phillips1,2,3 , N. Matin4 , B. Frias1 , M. M. Z. Zheng1,2 , M. Jia1 , C. West1 , A. M. Dorrance4 , I. Laher5 and A. V. Krassioukov1,6,7 1

International Collaboration on Repair Discoveries, Faculty of Medicine, University of British Columbia, Vancouver, Canada Experimental Medicine Program, Faculty of Medicine, University of British Columbia, Vancouver, Canada 3 Centre for Heart, Lung, and Vascular Health, Faculty of Health and Social Development, University of British Columbia, Vancouver, Canada 4 Pharmacology, Michigan State University, East Lansing, MI, USA 5 Deptartment of Pharmacology and Therapeutic, Faculty of Medicine, University of British Columbia, Vancouver, Canada 6 GF Strong Rehabilitation Center, Vancouver Coastal Health, Vancouver, Canada 7 Division of Physical Medicine and Rehabilitation, Department of Medicine, University of British Columbia, Vancouver, Canada

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Key Points

r Spinal cord injury (SCI) is associated with a 3–4 fold increased risk of stroke, and impaired r r r r

cerebral blood flow regulation, although the effect of SCI on the structure and function of the cerebral arteries is unclear. Using pressure myography to assess isolated vessels distended at physiological pressures, we provide novel evidence that experimental SCI leads to inward cerebrovascular remodelling, increased stiffness and impaired reactivity of the largest cerebral artery. Histochemical analyses revealed that a profibrotic environment within the largest cerebral artery occurs after SCI, which was characterized by greater collagen and less elastin. This may be due to increased transforming growth factor β, a well-known profibrotic signalling protein. Further analysis revealed that profibrotic alterations were not due to disruption of descending sympathetic pathways to the cerebrovasculature. Experimental SCI exerts a deleterious influence on the structure and function of cerebral arteries, which may underlie the increased risk of stroke and impaired cerebral blood flow regulation.

Abstract High-thoracic or cervical spinal cord injury (SCI) is associated with several critical clinical conditions related to impaired cerebrovascular health, including: 300–400% increased risk of stroke, cognitive decline and diminished cerebral blood flow regulation. The purpose of this study was to examine the influence of high-thoracic (T3 spinal segment) SCI on cerebrovascular structure and function, as well as molecular markers of profibrosis. Seven weeks after complete T3 spinal cord transection (T3-SCI, n = 15) or sham injury (Sham, n = 10), rats were sacrificed for either middle cerebral artery (MCA) structure and function assessments via ex vivo pressure myography, or immunohistochemical analyses. Myogenic tone was unchanged, but over a range of transmural pressures, inward remodelling occurred after T3-SCI with a 40% reduction in distensibility (both P < 0.05), and a 33% reduction in vasoconstrictive reactivity to 5-HT trending toward significance (P = 0.09). After T3-SCI, the MCA had more collagen I (42%), collagen III (24%), transforming growth factor β (47%) and angiotensin II receptor type 2 (132%), 27% less elastin as well as concurrent increased wall thickness and reduced lumen diameter (all P < 0.05). Sympathetic innervation (tyrosine hydroxylase-positive axon density) and endothelium-dependent dilatation (carbachol) of the MCA were not different between groups. This study demonstrates profibrosis and hypertrophic inward remodelling

C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

DOI: 10.1113/JP270925

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within the largest cerebral artery after high-thoracic SCI, leading to increased stiffness and possibly impaired reactivity. These deleterious adaptations would substantially undermine the capacity for regulation of cerebral blood flow and probably underlie several cerebrovascular clinical conditions in the SCI population. (Received 12 May 2015; accepted after revision 18 November 2015; first published online 4 December 2015) Corresponding author A. Krassioukov: ICORD-BSCC, University of British Columbia, 818 West 10th Avenue, Vancouver, BC, Canada, V5Z1M9. Email: [email protected] Abbreviations 5-HT, serotonin; AT1 , angiontensin II receptor type 1; AT2 , angiontensin II receptor type 2; MCA, middle cerebral artery; RAS, renin–angiotensin system; SCI, spinal cord injury; SMA, smooth muscle actin; TGF-β, transforming growth factor β; TH, tyrosine hydroxylase.

Introduction Over the past two decades, we have come to appreciate that cardiovascular disease is a primary cause of morbidity and mortality after spinal cord injury (SCI) (DeVivo et al. 2002; Garshick et al. 2005). Although a great deal of insight has been provided over that time illustrating cardiac as well as systemic vascular decline in the SCI population, there is scant literature regarding cerebrovascular function, and the associated consequences, in this population. Understanding cerebrovascular health after SCI is critical. Those with high-thoracic or cervical SCI are 300–400% more likely to suffer stroke as compared to uninjured counterparts, even after controlling for major confounding risk factors (Wu et al. 2012; Cragg et al. 2013). In fact, those with SCI have a similarly elevated risk of stroke as able-bodied smokers do of suffering a myocardial infarction (Yusuf et al. 2004). Also, cognitive dysfunction is widespread after SCI, which is probably mediated in part by cerebrovascular impairments (Davidoff et al. 1990; Jegede et al. 2010; Wecht & Bauman, 2013; Phillips et al. 2014b). We have recently shown in two human clinical trials that SCI results in cerebrovascular dysfunction, which was only partially restored when increasing blood pressure to able-bodied normal levels, indicating other important factors are playing a significant role (Phillips et al. 2014a,b). A more direct analysis of potential additional factors contributing to impaired cerebrovascular function (e.g. remodelling, stiffness, endothelial function, vasoconstrictive reactivity, profibrosis) after SCI is required to clearly elucidate the pathophysiology underlying cerebrovascular events in this population. For example, arterial remodelling, such as increased collagen and decreased elastin in arterial walls, occurs in response to profibrotic signalling [i.e. transforming growth factor β (TGF-β)] in models of volume unloading, physical inactivity and increased engagement of the renin–angiotensin system (RAS) (Zhang et al. 2001; Satoh et al. 2001; Tuday et al. 2009; Alessandri et al. 2010; Sofronova et al. 2015). These models of dysfunction also lead to increased stiffness and alterations in arterial

responsiveness to vasoactive signals (Geary et al. 1998; Tuday et al. 2009; Sofronova et al. 2015). After SCI, there is a combination of volume unloading, physical inactivity and increased reliance of the RAS system, which therefore may predispose to diverse cerebrovascular dysfunctions (Wecht et al. 2005; Handrakis et al. 2009; Groothuis & Thijssen, 2010). A fastidious understanding of cerebrovascular health after SCI will help guide interventions focused on mitigating the significant stroke burden, cognitive deficits and dysfunctional cerebral blood flow regulation that are commonly identified in those living with SCI. To date, no direct experimental examinations of cerebrovascular structure and function after SCI have been made. Herein, we have performed the first direct assessment of cerebrovascular function and structure in SCI rodents including: ex vivo pressure myography for passive structure, vasoconstrictor sensitivity and endothelial dilatation, as well as histological techniques to provide insight into the mechanisms underlying declining vascular health (i.e. collagen/elastin/TGF-β, morphometrics). We hypothesized that high-thoracic SCI would lead to impaired cerebrovascular function and structure, which would be associated with a profibrotic environment in the middle cerebral artery (MCA). Methods Experiments were initially conducted on 25 male Wistar rats (age 9 weeks, mass 250–350 g; Harlan Laboratories, Indianapolis, IN, USA). Animals were assigned to either: sham injured control (Sham; n = 10) or T3 complete spinal cord transection groups (T3-SCI; n = 15). Seven weeks after T3-SCI, MCA passive structure, endothelial function and vasoconstrictive responsiveness were assessed using pressure myography, as this is of analogous duration to that which is considered ‘chronic’ in the clinical SCI population (Krassioukov & Claydon, 2006). Surgery and animal care

Surgery and animal care were conducted according to standard procedures in our laboratory (Alan et al. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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The brain after spinal cord injury

2010; Ramsey et al. 2010). Animals received prophylactic enrofloxacin [Baytril; 10 mg kg−1 , S.C., Associated Veterinary Purchasing (AVP), Langley, Canada] for 3 days prior to SCI surgery. Moreover, for 5 days prior to surgery T3-SCI animals were provided enhanced caloric provision (i.e. Ensure, extra fruit, cereal) to ensure survival during weight loss, secondary to recovery. As such, body weight was elevated in the T3-SCI group prior to surgery (272.6 ± 5.6 vs. 323.3 ± 3.9 g; P < 0.05). On the day of surgery, enrofloxacin (10 mg kg−1 , S.C.), buprenorphine (Temgesic; 0.02 mg kg−1 , S.C., McGill University) and ketoprofen (Anafen, 5 mg kg−1 , S.C., AVP) were administered pre-operatively. Rats were anaesthetized with ketamine hydrochloride (70 mg kg−1 , I.P.; Vetalar; AVP) and dexmedetomidine (0.25 mg kg−1 , I.P.; Domitor; AVP). A dorsal midline incision was made in the superficial muscle overlying the C7–T3 vertebrae. The dura was opened at the T2–T3 intervertebral gap and the spinal cord was completely transected at the caudal portion of the intervertebral gap using microscissors. Complete transection was confirmed by two surgeons via visual separation of the rostral and caudal spinal cord stumps, and Gelfoam (Pharmacia & Upjohn Company, Pfizer, New York, USA) was placed between the stumps to achieve haemostasis. The muscle and skin were closed with 4–0 Vicryl and 4–0 Prolene sutures, respectively. Animals received warmed lactated Ringer’s solution (5 ml, S.C.) and recovered in a temperature-controlled environment (Animal Intensive Care Unit, Los Angeles, CA, USA), after being administered atipamezole (1 mg kg−1 , S.C., AVP) for reversal of dexmedetomidine. Enrofloxacin (10 mg kg−1 , S.C.), buprenorphine (0.02 mg kg−1 , S.C.) and ketoprofen (5 mg kg−1 , S.C.) were administered once a day for 3 days post-operatively. The bladder was manually expressed three to four times daily until spontaneous voiding returned (about 10 days after injury). All assessments and analyses were completed blinded of each condition/ group. Pressure myography studies. Seven weeks after injury, Sham and T3-SCI rats were anaesthetized with 3% isoflurane and sacrificed by decapitation after exsanguination for ex vivo pressure myography studies. MCA function and structure were assessed by pressure myography (Living Systems, St. Albans, VT, USA) as described previously (Pires et al. 2011). A branch-free segment of the MCA was isolated and mounted between two glass cannulas in a pressure myograph chamber, which was then placed in an inverted microscope coupled to a camera (Olympus SZ30, Centre Valley, PA, USA) and autodetection unit (Living Systems, VDA-10) under a 10× objective (Nikon E Plan LWD, 10×/0.25). We analysed MCA spontaneous myogenic tone generation and structural and mechanical properties. A 5 × 3 mm section of brain tissue containing the MCA was removed and placed in Ca2+ -free C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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physiological salt solution (PSS) at 4°C, with 1% BSA. A branch-free segment of the MCA was mounted on two glass micropipettes in the pressure myograph. MCAs were equilibrated at 80 mmHg in PSS containing (in mmol) 141.9 NaCl, 4.7 KCl, 1.12 KH2 PO4 , 1.7 MgSO4 .7H2 O, 2.8 CaCl2 , 10 Hepes, 5 dextrose and 0.5 EDTA (pH 7.4) until development and maintenance of at least 20% spontaneous myogenic tone, which was calculated using the following formula: %tone = [1−(active lumen diameter/passive lumen diameter)] × 100. The ability of the MCA to maintain a stable increase in external diameter with each pressure increment, and the generation of 20% tone ascertains the absence of leaks. Passive structure was assessed in Ca2+ -free PSS containing 0.002 M EGTA and intraluminal pressure was increased from 3 to 180 mmHg in 20 mmHg increments. Wall-to-lumen ratio and circumferential wall stress (Baumbach & Hajdu, 1993) as well as passive distensibility were calculated as described previously (Chan et al. 2010). The elastic modulus (β-coefficient) was calculated from the stress/strain curves using an exponential model (y = aeβx ), where β is the slope of the curve and is directly correlated to vascular stiffness. Reactivity of the MCA. Endothelial function in the MCA

was assessed by extraluminal perfusion of carbachol in a pressure myograph as per convention and previously described (Brueggemann et al. 2009; Wang et al. 2012). The MCA was then bathed in PSS pressurized at 80 mmHg to generate spontaneous myogenic tone. Increasing concentrations of carbachol (10−10 to 10−5 M) were then added to the bath. Carbachol, along with being a potent agonist of endothelial muscarinic receptors expressed in the MCA, cannot be degraded by acetylcholinesterase (Dauphin & Hamel, 1990). Furthermore, the low molecular weight of carbachol (182.696 g mol−1 ) favours a high capacity for migration through smooth muscle to activate the endothelial cells. After a thorough wash with calcium PSS, a single dose of 5-HT (serotonin; H9523; Sigma, St Loius, MO, USA) was added (10−5 M) to the bath. Immunohistochemical analyses Tissue collection and immunofluorescence. Animals were

sacrificed with an overdose of chloral hydrate (1 g kg−1 , I.P.) and the MCA was collected and post-fixed in 4% paraformaldehyde (PF), and then stored in 20% sucrose. A segment of the MCA from each animal of different experimental groups was embedded in optimal cutting temperature (OCT) freezing medium. Transverse 10 μm sections of the MCAs were cut on a cryostat microtome, collected in Superfrost Plus slides and stored at −80°C until further processing. The morphological assessment of MCAs was determined by immunofluorescence using the primary antibodies: mouse α TGFβ1 (1:200, Abcam,

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Cambridge, MA, USA; ab64715), rabbit α collagen (1:1000, Abcam, ab292), mouse α collagen 3 (1:300, Abcam, ab6310), mouse α angiotensin II receptor 1 (AT1 ; 1:200, Abcam, ab9391), rabbit α angiotensin II receptor 2 (AT2 ; 1:600, Abcam, ab19134), rabbit α elastin (1:400, Millipore, Billerica, MA, USA, AB2039) and rabbit α smooth muscle actin (SMA) (1:400, Abcam, ab5694). The MCA sections were thoroughly washed in PBS and 0.1 M PBS containing 0.3% Triton X-100 (PBST), followed by 1 h of incubation in 10% normal donkey serum in PBST. Sections were then incubated with the primary antibodies for 24 h at room temperature. Subsequently, sections were washed in PBST and incubated accordingly with Alexa-fluor 488 donkey anti-mouse (1:1000; Molecular Probes, Carlsbad, CA, USA) or donkey anti-rabbit conjugated Cy3 (1:1000; Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. Finally, all the sections were washed, mounted with Prolong Gold mounting medium (Molecular Probes) and observed in an AxioImager.M2 Zeiss microscope (HR R3; objective magnification 20×, ocular magnification 10×) using Zen 2 Pro. Objective: Zeiss PLAN NEOFLUAR 20×/0.50. Cy3, excitation peak = 550 μm, emission peak = 570 μm, tetramethylrhodamine filter settings. Alex Fluor 488 excitation peak = 495 μm, emission peak = 519 μm, fluorescein isothiocyanate filter settings. Resolution of collected images: 942,490,000 pixels cm–2 (i.e. 3.070 pixels μm–1 ).

TH analysis of MCA whole mounts. A small segment of the MCA vessel was used to determine TH expression (1:1000; anti-rabbit; Millipore; ref.: AB152, USA) by immunofluorescence using Cy3 secondary antibody, and therefore TH+ fibre expression. For this, the vessel was placed in a small Eppendorf tube and the immunoreaction proceeded as described above. Finally, the vessel was placed in a glass microscope slide, mounted with Prolong Gold mounting medium (Molecular Probes) and viewed in the microscope.

Quantification

Analysis of TH innervation in MCA whole mounts.

The intensity of immunoreactivity of morphological markers of the MCAs was assessed using Fiji Software (based on ImageJ, http://fiji.sc/Downloads#Fiji). For all primary antibodies except tyrosine hydroxylase (TH), staining intensity was averaged from four sections per animal selected from the middle third of the MCA. We generated a region of interest using the advential border of the artery and the lumenal interface as the outer and inner boundaries. Within this region of interest, relative intensity was quantified. We were careful to obviate the surrounding connective tissue from within the region of interest. In all cases, a reference intensity of unstained tissue was measured to determine background intensity, which was deducted from the average intensity of each section to calculate the mean net staining intensity. Furthermore, a negative control (i.e. only secondary antibody added in the absence of primary antibody) was required for each protocol to ensure specificity of secondary to primary antibody (Frias et al. 2015). In addition, the area of the region of interest was normalized in every tissue section. Staining intensities determined in Sham were used as standardizing controls.

Immunohistochemical morphometric assessment of the MCA

Using the SMA staining of the MCA, arterial wall thickness and lumen diameter were measured using Image J. For each section of an artery, four sites were selected representing each quadrant of the vessel, and a line was drawn across the span of staining perpendicular to the tangent line, and the length of this line was measured. The average of the four measurements was used to denote wall thickness for each section. Lumen diameter was measured by taking the average of the widest and the narrowest part of the lumen. Four sections were measured and averaged from each animal. Considering the emission peak of the secondary antibody (Cy3) used with the SMA primary antibody and numerical aperture of the microscope, our resolution limit for this assessment was 570 μm, and data were reported to the nearest whole number.

Quantification was carried out manually on Z-stack images from the MCAs of each animal using Fiji Software. A rectangular grid was divided into equal squares and was placed over the region of interest on each of the upper and lower planes of the vessel. A grid was superimposed over the entire imaging frame (44 μm2 per grid sector). From the grid, a 5 × 5 sector region of interest was selected (220 μm2 ), with the inclusion criteria that the entire region of interest is within the boundary of the artery. The region of grid and region of interest were chosen randomly between animals. The quantification of the fluorescence intensity and innervation density was conducted within this region. A measurement was obtained for longitudinal and circumferential density by counting the number of horizontal and vertical grid line crosses, respectively. A value for total innervation density (longitudinal + circumferential) was then calculated for the vessel region and compared across treatments (Hesp et al. 2012). Statistical analyses. Body mass, brain mass, brain mass/body mass, spontaneous myogenic tone generation, 5-HT responsiveness, passive structure at 80 mmHg and C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society


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immunohistochemistry data were analysed by Student’s t test or a non-parametric alternative when the data did not fit a normal distribution model. Repeated measures ANOVA was also used for passive structure over increasing transmural pressures using pressure myography (time × group). Concentration–response curves for carbachol were further analysed by non-linear regression (Hill equation) to calculate logEC50 and logECmax . All statistical analyses were carried out using GraphPad Prism 6.0 software (GraphPad, San Diego, CA, USA). Differences between means were considered statistically significant at P < 0.05. Data are shown as mean ± SEM.

Results Physiological parameters

At the end point of the experimental protocol, body mass (T3-SCI: 315 ± 11 vs. Sham: 344 ± 5 g: P = 0.06), brain mass (T3-SCI: 2.2 ± 0.04 vs. Sham: 2.2 ± 0.03 g: P = 0.85) and brain mass/body mass ratio (Sham: 0.63 ± 0.01 vs. T3-SCI: 0.69 ± 0.03; P = 0.20) were not different between groups.

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SCI detrimentally altered structure of the MCA

After T3-SCI, pressure myography demonstrated that animals experienced 10% reduced MCA lumen diameter (P < 0.05, Fig. 1A and B). The MCA lumen cross-sectional area was also reduced by 20% after T3-SCI (P < 0.05), without concurrent changes in wall thickness or wall cross-sectional area (Table 1), leading to a 14% greater wall-to-lumen ratio after T3-SCI (P < 0.05, Table 1). Morphometrics ascertained using immunohistochemistry also showed reduced lumen diameter, and increased wall-to-lumen ratio after T3-SCI, although this technique also demonstrated a 42% increased MCA wall thickness in the T3-SCI cohort (P < 0.05, Fig. 2). Stiffness was also increased substantially after T3-SCI. Specifically, distensibility was reduced 40% after T3-SCI (P < 0.05, Fig. 3). Furthermore, a 40% reduction in vessel strain and a 10% reduction in vessel stress (both P < 0.05; Table 1) led to a 36% increase in β stiffness trending towards significance (P = 0.09, Fig. 3). SCI did not change myogenic reactivity of the MCA but reduced MCA contractility. Spontaneously generated

myogenic tone was not different between groups in

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Figure 1. Complete T3-SCI led to inward remodelling of the MCA Pressure myography demonstrated complete T3-SCI (n = 7) led to inward remodelling of the MCA (D), characterized by a decrease in lumen size (A and B), but no corresponding increase in wall thickness was detected using repeated measures (C). Comparisons were made using only physiological intraluminal pressures (40–180 mmHg). ∗ Significantly different from Sham (n = 5; P < 0.05). Repeated measures ANOVA (main effect). C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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Table 1. Ex vivo pressure myography data showing remodelling of the MCA after T3 spinal cord injury Sham Lumen diameter (μm) Outer diameter (μm) Wall thickness (μm) Wall to lumen ratio Lumen CSA (μm2 ) Vessel CSA (μm2 ) Wall CSA (μm2 ) Vessel strain Vessel stress Distensibility

297 331 17 0.057 69,620 86,460 16,840 0.7333 700 73

± ± ± ± ± ± ± ± ± ±

10 11 1 0.001 4,867 5952 1141 0.15 15 15

SCI 265 299 17 0.065 55,410 71,660 16,250 0.4400 625 44

± ± ± ± ± ± ± ± ± ±

5∗ 5∗ 1 0.001∗ 2217∗ 2768∗ 1016 0.05∗ 25∗ 5∗

Values are mean ± SEM at an intraluminal pressure of 80 mmHg. CSA, cross-sectional area. ∗ Significantly different from Sham (P < 0.05).

absolute terms (Baseline: Sham = 308.2 ± 12.3 vs. T3-SCI = 280.2 ± 22.4 μm; Post-Myogenic Tone: Sham = 199 ± 36 vs. T3-SCI = 184 ± 32 μm; P = 0.86), or as a per cent (Sham = 36 ± 4; T3-SCI = 35 ± 3%; P = 0.87). We did observe a trend for a reduced contractile response to 10−5 M 5-HT which was on average 33% lower in T3-SCI (P = 0.09; Fig. 3D). SCI did not impair endothelial function in the MCA. After T3-SCI, there was no difference in MCA logEC50 or logECmax (Fig. 4). Specifically, endothelial function of the MCA at 80 mmHg was not significantly different between groups. Neither maximal dilatation to carbachol (logECmax ) or sensitivity (logEC50 ) was different between Sham and T3-SCI. SCI-mediated profibrosis within the MCA. After T3-SCI,

there was 42% increase in collagen I and a 24% increase in collagen III expression within the MCA coinciding with a 47% increase in TGF-β (P = 0.04, P = 0.005 and

P = 0.02, respectively; Fig. 5). Elastin was reduced 27%, while angiotensin II receptor type 2 was increased 132% (P = 0.04 and P < 0.001, respectively; Fig. 5). Figure 5C shows for representative staining for each primary antibody. Complete SCI at the T3 level did not reduce sympathetic innervation of the MCA. After the present model of T3

complete T3-SCI, no reduction in TH+ axonal density or fluorescence intensity occurred (Fig. 6). Discussion This is the first study indicating that high-thoracic SCI leads to extensive cerebrovascular maladaptation, including hypertrophic inward remodelling, stiffening and possibly impaired reactivity. We also demonstrate that the cerebrovascular environment is profibrotic after high-thoracic SCI, characterized by increased collagen, decreased elastin and increased expression of TGF-β, a protein involved in the signalling cascade leading to fibrosis. As sympathetic pathways to the brain vasculature were not disrupted in the present T3 transection model, it is our contention that these deleterious alterations in the cerebrovasculature are a secondary consequence of a combination of lower capacity for physical exercise, reduced alterations in blood volume/pressure after T3-SCI and an over-reliance on the RAS (Noreau et al. 1993; Wecht et al. 2009). These factors have each been shown to induce profibrosis (Leask & Abraham, 2004; Duprez, 2006; Marchesi et al. 2008; Tuday et al. 2009; Sofronova et al. 2015). The present findings provide evidence demonstrating advanced arteriosclerotic progression in the brain after T3-SCI and insight into the underlying mechanisms. Taken together, a profibrotic environment within the cerebrovasculature after T3-SCI probably plays a role in a variety of cerebrovascular dysfunctions after SCI. These include the increased risk of stroke, as well as other

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Figure 2. T3-SCI results in hypertrophic inward remodelling of the MCA Morphometric analysis of smooth muscle actin immunohistochemical staining demonstrated evidence that T3-SCI results in hypertrophic inward remodelling of the MCA, as evidenced by greater wall thickness (A), reduced lumen diameter (B) and increased wall-to-lumen ratio (C) in the T3-SCI group. ∗ Significantly different from Sham (P < 0.05). Unpaired t test. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society


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cerebrovascular conditions such as dysfunctional cerebral blood flow regulation and vascular cognitive impairments (Cragg et al. 2013; Wiesmann et al. 2013; Phillips et al. 2014a,b), and as well may lead to reduced global cerebral blood flow (Phillips et al. 2013a). Remodelling of the MCA has been well documented in a variety of clinical conditions (Pires et al. 2013; Dorrance et al. 2014). After T3-SCI, the present study illustrated a decrease in MCA lumen diameter, increased wall thickness and a significant increase in wall-to-lumen ratio. Increased MCA stiffness in the present T3-SCI cohort demonstrates that cerebrovascular remodelling after T3-SCI is probably directly impacting the distensibility of this vessel. Mechanistically, the reduced distensibility would be expected considering the corresponding immunohistochemical assessments demonstrating increased collagen and decreased elastin expression in the MCA after T3-SCI (Izzard et al. 2006). Previous research has implicated the loss of suprapinal sympathetic tone as a mechanism leading to arterial remodelling. Indeed, in a rabbit model, increased collagen A

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and decreased elastin were shown in the aorta after chronic sympathectomy (Fronek et al. 1978). It is unlikely we disrupted descending sympathetic pathways of supraspinal origin in the present study, however, as we have shown that sympathetic fibre density of the MCA is identical between the two groups, and would have been decreased or absent if the sympathetic pathway was disrupted (Hesp et al. 2012). There are reductions in physical activity and blood volume/pressure after cervical and high-thoracic SCI (Scott et al. 2011), and we have previously reported that blood pressure is significantly reduced following T3 spinal cord transection in Wistar rats (West et al. 2015). The combination of these factors probably exacerbates large artery dysfunction in our model of T3 complete SCI, as reduced physical activity and blood volume/pressure have both been implicated in the noted large artery dysfunction (Geary et al. 1998; Zhang et al. 2001; Tuday et al. 2009). These studies reported similar but not identical findings to the present study, where profibrosis occurred and vasoreactivity of the cerebrovasculature was impaired after B

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Figure 3. Mechanical properties of the MCA after T3-SCI A–C, mechanical properties of the MCA were deleteriously influenced by T3 complete SCI (n = 7) resulting in reduced distensibility (A), impaired stress–strain relationship (B) and a trend for increased β-stiffness (C). D, MCA constrictive responses to 10−5 M 5-HT. Spinal cord transection at the T3 level (n = 7) led to a trend indicating impaired vascular responsiveness compared to Sham-injured animals (Sham; n = 5). ∗ Significantly different from Sham (n = 5; P < 0.05). A and B: repeated measures ANOVA (main effect). C and D: unpaired t test. C 2015 The Authors. The Journal of Physiology C 2015 The Physiological Society

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exposure to reductions in physical activity, and reduced blood volume. It is interesting to note that one of the mentioned studies showed a counterintuitive increase in vasoreactivity of the basilar artery, although this may be due to regional vasoreactivity differences within the cerebrovasculature as has been previously reported (Sato et al. 2012; Phillips et al. 2014a). In addition to reduced exercise capacity and autonomic instability, after cervical and high-thoracic SCI the

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cardiovascular system also adapts to rely predominantly on the RAS for blood pressure maintenance (Wecht et al. 2005; Handrakis et al. 2009; Groothuis & Thijssen, 2010). Increased RAS leads to a profibrotic environment, which is characterized by excessive production, deposition and contraction of the extracellular matrix (Leask & Abraham, 2004). The protein TGF-β is a major profibrotic signalling cytokine that is upregulated by elevated RAS activity (Rosendorff, 1996). As evidenced by our T3-SCI

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Figure 4. Endothelial function of the MCA after SCI Endothelial function of the MCA, assessed at 80 mmHg with carbachol, was not significantly impacted by SCI. Specifically, neither maximal dilatation to carbachol (logECmax ) nor sensitivity (logEC50 ) was different between Sham (n = 5) and T3 complete SCI animals (n = 6). One-way ANOVA.

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Figure 5. Profibrotic middle cerebral artery after SCI A, immunofluorescence intensity of proteins associated with profibrosis between Sham and T3-SCI. Values are mean ± SEM. TGF-β, transforming growth factor beta; AT1 , angiotensin II receptor type 1; AT2 angiotensin II receptor type 2; SMA, smooth muscle actin. ∗ Significantly different from Sham (P < 0.05). Unpaired t test. B, selection of the region of interest (ROI) for quantification of staining intensity used the adventitial layer as the outer boundary and the medial border of the internal elastic lamina (IEL) as the inner boundary. C, example immunohistochemical images at 20× magnification from Sham (middle) and SCI animals (right) for: CI, collagen 1; CIII, collagen 3; TGFβ, transforming growth factor beta. Inset: example staining for each primary antibody at 63× magnification.


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animals, SCI results in elevated collagen content and increased TGF-β. It is worth noting that TGF-β is typically considered an up-regulator of elastin expression, although increases in RAS activity have also been shown to lead to elastin degradation (Pons et al. 2011). It appears in the present study that the latter elastin degradation trumped the upregulation expected from increased TGF-β expression. Also, only AT2 receptors were increased after T3-SCI in the present study, although this may have occurred as an adaptation to counteract the profibrotic status induced by increased RAS activation of the AT1 (Montezano et al. 2014). Accordingly, it is fair to posit that after T3-SCI profibrosis develops within the large cerebral arteries (probably due to increased RAS activity) leading to overexpression and contraction of the extracellular matrix, resulting in reduced distensibility and hypertrophic remodelling. After T3-SCI, reduced lumen diameter, increased stiffness and ensuing increased vascular resistance of the MCA likely leads to the frequent observation of reduced cerebral blood flow (Phillips et al. 2013a), which would exacerbate disorders associated with cerebral hypoperfusion, including vascular dementia, Alzheimer’s disease and recovery from ischaemic stroke (Vorstrup et al. 1986; Farkas & Luiten, 2001; Roher et al. 2012). Furthermore, increased fibrosis within the cerebrovasculature is probably contributing to dysfunctional cerebrovascular regulation after cervical and high-thoracic SCI (e.g. impaired neurovascular coupling, cerebrovascular reactivity) (Wilson et al. 2010; Phillips et al. 2013b). Endothelial function of the MCA was not diminished after T3-SCI, which is in agreement with several studies

showing normal endothelial function in vasculature rostral to the lesion level after SCI (de Groot et al. 2004, 2006). Considering the already elevated risk of stroke in this population, any decrease in endothelial function would further exacerbate this risk by inhibiting the capacity to circumvent occlusion through collateral artery dilatation (Coyle, 1987). There was a trend suggesting a reduction in vascular reactivity to 5-HT after T3-SCI in the present study. Although this could be attributed to increased profibrosis and stiffness of the MCA, the fact that endothelial responsiveness was not also impaired suggests that factors other than increased stiffness of the MCA could be responsible. One possibility is that an increase in sympathetic nervous system activity above the lesion level (i.e. from the T1–2 levels) after T3 complete SCI impacted reactivity. It well documented that sympathetic nervous system activity is inversely associated with vascular constrictive sensitivity (Brock et al. 2006; Charkoudian et al. 2006), and we have shown that after high-thoracic SCI, there are indications of increased sympathetic activity to target organs above the lesion level (Claydon & Krassioukov, 2008; Lujan et al. 2010, 2012), which would be expected to lead to vasoconstrictor hyposensitivity rostral to injury. From a clinical perspective, impaired vasoconstrictor function of the MCA would certainly inhibit functional cerebral autoregulation, particularly during periods of hypertension, such as autonomic dysreflexia, which are well reported to lead to cerebral haemorrhage (Wan & Krassioukov, 2014). There are a number of exciting directions for future research, after illustrating for the first time impaired

TH+ axon density (crosses 220 μm–2)

Sham

Figure 6. TH+ axonal density and fluorescence intensity after T3-SCI Top: representative immunohistochemical images from Sham (left; n = 3) and T3-SCI animals (right; n = 8) for tyrosine hydroxylase (TH). Bottom left: TH immunoreactive (TH-ir) relative axon density of the MCA. Bottom right: white arrows denote which axons were considered TH positive. No differences occurred between Sham injured and animals with T3 complete spinal cord injury. Unpaired t test.

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150

100

100 μm

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0 Sham

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cerebrovascular structure and function after experimental T3-SCI. First, the mechanism underlying the profibrotic environment in the vasculature after T3-SCI needs to be elucidated. An initial step would be to assess the effect of AT1 /AT2 receptor blockade/exercise training after high-thoracic SCI, with a particular focus on matrix metalloproteinases known to impact collagen and/or elastin (Zieman et al. 2005). Another necessary step is to understand the role autonomic dysreflexia (i.e. transient hypertensive episodes that occur on a daily basis after high-thoracic or cervical SCI) plays in cerebrovascular health in this population (Hubli et al. 2015). Moreover, understanding how profibrotic cerebrovascular remodelling affects cerebral blood flow regulation in this model would be a logical extension of the present study. We did not assess behaviour outcomes related to cerebrovascular dysfunction secondary to T3-SCI, such as cognitive function. It would be ideal to assess cognitive function after high-thoracic SCI as compared to Sham. Given the significant motor dysfunctions of the hind limbs after SCI, however, it is difficult to truly assess cognitive function through standard protocols such as the Morris water maze. We are certain that dilatation after carbachol administration in the present study was due to endothelial activation, although it is possible that greater magnitude dilatation would have occurred if carbachol was administered intralumenally. It is also possible that greater between-group differences would have occurred for 5-HT if a range of doses were used. Different vessel preparations (i.e. ex vivo living tissue vs. post-fixed frozen tissue) may have led to the discrepancy in absolute wall thickness between pressure myography and immunohistochemistry staining. Immunohistochemistry was used to indirectly quantify protein expression in the present study. Although clinically this technique is not considered quantitative due to a lack of reference standards, immunohistochemistry, when applied using identical filtering and staining protocols as well as ensuring a dark negative control, can provide accurate quantification of protein expression (Taylor & Levenson, 2006; Frias et al. 2015). Conclusions Structure and function of the cerebrovasculature are deleteriously impacted by high-thoracic or cervical SCI, secondary to the development of a profibrotic environment. This profibrosis may be the consequence of a combination of an increased influence of RAS, reduced physical activity and reductions in blood pressure/total blood volume in this model of SCI, but not the direct impact of decentralized sympathetic pathways to the brain. Starkly increased risk of stroke, dysfunctional cerebral blood flow regulation and vascular cognitive impairments may all be associated with impaired large cerebral artery

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Additional information Competing interests None. Author contributions Experiments were performed in the laboratory of A.V.K. Contributions were as follows: (1) conception and design of the experiments; (2) collection, assembly, analysis and interpretation of data; (3) drafting the article or revising it critically for important intellectual content. A.A.P.1,2,3 , N.M.1,2,3 , B.F.1,2,3 , M.M.Z.Z.1,2,3 , M.J.1,2,3 , C.W.1,2,3 , A.M.D.1,2, 3 , I.L.1,2,3 , A.V.K.1,2,3 Funding A.A.P. is funded by the Heart and Stroke Foundation of Canada (HSFC), the Michael Smith Foundation for Health Research and the Craig H. Neilsen Foundation. A.V.K. is funded by the Canadian Institute for Health Research, Rick Hansen Institute, the Craig Neilson Foundation, Christopher and Dana Reeves Foundation, and the HSFC. Acknowledgements Paul Lesack (Data/GIS Analyst, University of British Columbia) is acknowledged for his remarkable artistic contributions and technical expertise in fulfiling our vision for the cover art associated with this manuscript: [email protected]. Dr Matthew Ramer is acknowledged for his extensive consultation regarding immunohistochemistry protocols.
