Vascular-Cognitive Impairment following High-Thoracic Spinal Cord Injury Is Associated with Structural and Functional Maladaptations in Cerebrovasculature

Experimental design

All experimental procedures were carried out in accordance with the Guide for the Care and Use of Experimental Animals established by the Canadian Council on Animal Care and were approved by the University of British Columbia Animal Care Committee (approval certificate number: A18-0183). Twenty-eight adult (250–300 g) male Wistar rats were assigned into either a naïve control (CON, n = 14) or T3-complete spinal cord transection group (SCI, n = 14). Fourteen weeks post-injury, all rats were assessed for short-term memory using a novel object recognition test (NORT, Fig. 1). Following the NORT, the groups were subdivided equally (n = 7 each). Each subgroup was assigned for either in vitro physiological assessments using pressure myography or for magnetic resonance imaging (MRI) followed by immunohistochemistry.

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FIG. 1.

Experimental timeline. The rats were divided into two experimental groups (n = 14 each): CON (uninjured) and SCI (complete transection at T3 spinal segment). At 14 weeks post-SCI (and age-matched controls), all rats underwent NORT. Following NORT, the groups were subdivided equally and assigned to: 1) in vitro physiological assessments using pressure myography, and 2) MRI followed by immunohistochemistry. MRI, magnetic resonance imaging; NORT, novel object recognition test; SCI, spinal cord injury. Color image is available online.

T3 Transection surgery and animal care

Surgery and animal care were conducted as described previously. ⁹ Rats were pre-treated with enrofloxacin (Baytril; 10 mg/kg, s.c., Associated Veterinary Purchasing [AVP], Langley, British Columbia, Canada) for 3 days. The animals were fasted the night before surgery. On the day of surgery, anesthesia was induced with 5% isoflurane (AErrane; AVP) in 100% oxygen at a flow rate of 1 L/min and maintained at 2–3% during the surgery. Enrofloxacin (10 mg/kg, s.c.), buprenorphine (Temgesic; 0.02 mg/kg, s.c., University of McGill Animal Resources Centre), and lactated Ringer's solution (5 mL, s.c.) were administered pre-operatively.

A dorsal midline incision was made through the skin and superficial muscle layer, as well as a blunt dissection of deep muscles overlying T1-4 vertebrae. Deep muscles around T2-3 vertebrae were carefully dissected to expose the intervertebral gap. The dura at the gap was then opened, and the spinal cord was transected with microscissors. Complete transection was confirmed by two surgeons via visual separation of the rostral and caudal spinal cord stumps under the microscope. Absorption sponges and Gelfoam (Pharmacia & Upjohn Company, Pfizer, New York, USA) were used to achieve hemostasis. The muscle and skin of the surgical site were closed with 4–0 Vicryl and 4–0 Prolene sutures, respectively. Animals received warmed lactated Ringer's solution (5 mL, s.c.) and were allowed to recover from anesthesia in a temperature-controlled environment (Animal Intensive Care Unit, HotSpot for Birds, Los Angeles, CA, USA). Enrofloxacin (10 mg/kg, s.c., once per day) and buprenorphine (0.02 mg/kg, s.c., twice per day) were administered for 3 days post-surgery.

Animals with SCI were housed in specialized cages, with rubber matting to facilitate movement, low-reaching water bottles, and food scattered on the cage bottom to encourage foraging. Animals were supported with an enriched diet, including meal replacement shake (Ensure, Abbott Laboratories, Abbott Park, IL, USA), fruit, spinach, HydroGel (Clear H₂O, Westbrook, ME, USA), peanuts, and kibble (LabDiet, Rodent Diet 5001; PMI International, St. Louis, MO, USA). The bladder was manually emptied three times daily until spontaneous bladder function returned (∼10 days after injury). Health status was monitored and recorded daily for the first 2 weeks after surgery and on alternative days thereafter. All experiments and analyses were performed with the investigators completely blinded of the group assignments.

NORT

Short-term memory was evaluated using the NORT, a widely implemented test to assess retention memory and interest in exploring novel objects. ¹⁰ One day prior to the testing session, animals were individually placed in an empty container for 1 h to encourage habituation. One hour before the test, animals were exposed to two identical objects for 10 min in the same container, and then one object was replaced with a novel object. ^{11 –14} During the test, animals were allowed to explore the open field freely for 5 min while being videotaped. Discrimination index (D-index) was calculated by dividing the difference between the exploration time devoted to novel (TN) and Object 1 (T1) divided by the total exploration time (DI = [TN – T1]/[TN + T1]*100). ¹¹

CBF

CBF maps were acquired using arterial spin labeling (ASL) MRI as previously described. ¹⁵ Briefly, water molecules from inflowing blood were magnetically excited by a spin inversion. The overall magnetization of a downstream area of the brain was modified upon the arrival of these labeled molecules. The resultant signal describes the amount of arterial blood delivered to the imaging slice within the transit time of the experiment. On the day of imaging, rats were sedated with isoflurane and maintained on a ventilator (Harvard Apparatus Inspira ASV, USA) to maintain expired CO₂ at 35 mm Hg. Body temperature was maintained at 37°C. Assessments were performed on a 7 Tesla MRI scanner (Bruker Biospec 70/30 USR, Bruker, Ettlingen, Germany) using a quadrature volume transmit coil and 4-channel phased-array receivers.

Multiple inversion recovery echo-planar imaging (EPI) scans were acquired to obtain calibration data (tissue T1, inversion efficiency, and proton density) for the ASL scan (recovery time = 10 sec, echo time = 11.47 msec, 22 inversion times between 30 and 8000 msec spaced geometrically, field of view = 40 × 40 mm, matrix = 64 × 64, slice thickness = 2 mm, 9 slices, single shot). Flow-sensitive alternating inversion recovery EPI with a pre-saturation preparation ¹⁶ was used to acquire the ASL data at five inversion times to allow correction for variable transit times using same geometry as the calibration scans. CBF maps were calculated by fitting the full perfusion model, including non-zero arterial transit time and bolus outflow. ¹⁷

Pressure myography

MCA function was assessed using pressure myography (Living Systems, St. Albans, VT, USA) as described previously. ^9,18,19 A 5 × 3-mm section of brain tissue containing the MCA was removed and placed in Ca²⁺ free physiological salt solution (PSS) at 4°C, with 1% bovine serum albumin. A branch-free M2 segment of MCA was dissected. The M2 segment of MCA is the branch-free segment of the MCA and is a widely accepted segment for the study of pressure-diameter relationships because the other branched segments cannot hold the perfusion pressure. ^{20 –22} The vessels were mounted on two glass micropipettes in the pressure myograph, and the diameter was measured using a digital CCD camera. MCAs were equilibrated at 80 mm Hg for 45 min in PSS containing (in millimoles) 141.9 NaCl, 4.7 KCl, 1.12 KH₂PO₄, 1.7 MgSO₄.7H₂O, 2.8 CaCl₂, 10 HEPES, 5 dextrose, and 0.5 EDTA (pH = 7.4) solution. The baseline of the inner diameter of the MCA was measured, and only vessels that developed >20% spontaneous myogenic tone were used for the experiment. Passive distensibility was assessed in Ca^2+-free PSS containing 0.002 M EGTA as intraluminal pressure was increased from 20 to 180 mm Hg in 20 mm Hg increments.

Endothelial function was assessed by measuring the response to administration of carbachol in the bath as previously described. ^23,24 The MCA was first bathed in PSS and pressurized at 80 mm Hg to generate a stable degree of spontaneous myogenic tone. The vasodilator response to increasing concentrations of carbachol (10⁻¹⁰ to 10⁻⁵ M) was then determined. ²⁵ The vessel was pre-incubated with a transient receptor potential cation channel 4 (TRPV 4) antagonist, HC067047 (10⁻⁶ M), for 15 min and the responses to carbachol (10⁻¹⁰ to 10⁻⁵ M) were recorded to investigate the mechanism involved. ²⁶ Finally, the vessels were bathed in Ca²⁺-free PSS, and a passive pressure diameter curve was recorded. Myogenic tone was calculated using the following formula: % tone = (1−[active lumen diameter/passive lumen diameter]) × 100.

Perfusion and tissue collection

Animals were euthanized with isoflurane followed by intraperitoneal injection of chloral hydrate. The cerebral vasculature was fixed by transcardial perfusion with 100–150 mL of phosphate-buffered saline (PBS; at 80–90 mm Hg) and 400 mL of 4% paraformaldehyde at 120–140 mm Hg. The brain was harvested and placed in 4% paraformaldehyde for 24 h at 4°C and then transferred to 20% sucrose for 3 days. A 2- to 3-mm long segment of MCA was gently dissected from each hemisphere and used for histological analyses.

Immunohistochemistry

The M2 segments of MCA with a length of approximately 5 mm and nearly uniform cylindrical shape were isolated from the animals, and were embedded in Cryomatrix^®. We systematically randomly sampled a series of sections for stereology as previously described. ²¹ Using a cryostat, 10-μm frozen transverse sections were cut and were placed on Superfrost Plus slides. The sections were then randomly selected, and stored at −80°C until processed for immunohistochemistry.

Briefly, MCA sections were washed in PBS and 0.1 M PBS containing 0.3% Triton X-100 (PBST). They were blocked for 1 h in 10% normal donkey serum in PBST. Sections were then incubated with primary antibodies to: collagen 1 (COL 1, 1:500, Abcam, ab34710), collagen 3 (COL 3, 1:300, Abcam, ab6310), TRPV 4 (1:300, generous gift from Dr. Stefan Heller's lab at Stanford), cluster of differentiation 31 (CD31, 10 μg/mL, R&D systems, AF3628), or smooth muscle actin (SMA, 1:400, Abcam, ab5694) overnight at room temperature. On the second day, the sections were washed in PBST and incubated with species-specific secondary antibodies: Alexa™-fluor 488 donkey anti-mouse (1:1000, Molecular Probes^©, USA), Alexa™-fluor 488 donkey anti-goat (1:1000, Molecular Probes^©), or Cy3 conjugated donkey anti-rabbit IgG antibody (1:1000, Jackson ImmunoResearch) for 1 h. Finally, the sections were washed with PBST, coverslipped with Vectashield mounting medium (Vector Laboratories, Inc.), and images were captured using a confocal microscope with identical acquisition parameters.

Quantification

The intensity of immunoreactivity of MCAs was assessed using Fiji Software (http://fiji.sc/Downloads#Fiji). Staining intensities were averaged from four sections per animal. For the analyses of COL 1, COL 3, and SMA, the region of interest (ROI) were confined between the adventitial border of the artery and the lumenal interface. TRPV 4 and CD31 were quantified in the endothelial layer (Fig. 2). Further, a negative control consisting of staining with secondary antibody alone was performed for each antibody. ^9,27

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FIG. 2.

Carbachol-induced relaxation is diminished in MCA after chronic SCI, which is closely associated with the loss of TRPV 4 and CD31 staining in the endothelium. (A,B) Dose-response curve of MCA in control (CON, n = 6) and rats with SCI (SCI, n = 6) to carbachol. (C) Maximal dilation of MCAs to carbachol. (D) logEC₅₀ to carbachol. (E) MCA distensibility curve under Ca²⁺ free solution. (F) Representative images of transverse sections of the MCA immunostained for CD31 and TRPV 4 illustrating the co-localization of the fluorescent signals in the endothelium (arrows). (G) Comparison of the immunofluorescence staining of CD31 and TRPV 4 in the endothelial layer of MCAs in control and SCI rats. Values are mean ± standard error; A and B, ****significantly different from CON using a one-way ANOVA with Tukey's post hoc test (p < 0.0001); E and G, *significantly different from CON (p < 0.05) using an unpaired t test. ANOVA, analysis of variance; CD31, cluster of differentiation 31; MCA, middle cerebral artery; SCI, spinal cord injury; TRPV 4, transient receptor potential cation channel 4. Color image is available online.

Morphometric assessment of the MCA

The arterial wall thickness and lumen diameter were measured using the transverse sections of the MCA immunostained with SMA (four sections per rat). Within each section, four sites were selected, representing a quadrant of the vessel, and a line was drawn across the media of the vascular wall 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.

Tyrosine hydroxylase (TH) staining of the MCA whole mounts

A small segment (3–4 μm) from the middle third of the MCA was stained for tyrosine hydroxylase (TH, 1:1000, anti-rabbit, Millipore, AB152) to compare the degree of sympathetic innervation. The density of TH-positive (TH⁺) nerve fibers was measured using Fiji Software (using ImageJ, http://fiji.sc/Downloads#Fiji). A grid was generated over the entire image (44 um²/grid sector). A 5 × 5 ROI was randomly selected, and the intersections between the grid and the stained fibers were counted. The number of crossings of fibers with the grid was used to determine the density of TH⁺ fibers that innervate the middle third of the MCA as previously described. ²⁸

Statistical analysis

Mean values ± standard error are presented. The significance of differences between mean values in the control and spinal cord transected groups was determined using a Student's t test or a non-parametric alternative when the data were not normally distributed. Repeated measures analysis of variance (ANOVA) was used for the concentration-response curves for carbachol, which were further analyzed by non-linear regression (Hill equation) to calculate EC50 and Emax values. All the statistical analyses were carried out using GraphPad Prism 7.0 software (GraphPad, San Diego, CA). A p-value of less than 0.05 was considered to be statistically significant.

Altered endothelial function and mechanical properties of the MCA after chronic SCI

The endothelial function of the MCA was impaired in the rats with SCI (Fig. 3). The maximal vasodilation response to carbachol was 55% (SCI vs. CON: 13.4 ± 1.9% vs. 29.63 ± 2.8%, respectively) less in the SCI group than in the uninjured controls (Fig. 3A,B), but the EC50 was not significantly different (Fig. 3C,D). Further, when tested across a range of intraluminal pressures in Ca²⁺free solution, the distensibility of the MCA was significantly reduced, by about 37% (SCI vs. CON: 30.5 ± 2.9% vs. 42.0 ± 4.0%, respectively) over a range of pressures from 80 to 180 mm Hg (Fig. 3E).

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FIG. 3.

Chronic SCI leads to increased collagen deposition in the arterial wall. (A) Immunofluorescence intensity of proteins associated with fibrosis between control (CON) and SCI. Values are mean ± SEM. Intensity for COL 1 was increased by 50%. No change was seen in Col 3 and SMA. *Indicates significantly different from CON (p < 0.05); unpaired t test. (B) No morphological change was found in the MCA passive structure. (C) TH⁺ axon density does not change after chronic SCI. Arrows indicate TH⁺ axons. COL 1, collagen 1; MCA, middle cerebral artery; SCI, spinal cord injury; SEM, standard error of the mean; SMA, smooth muscle actin; TH⁺, tyrosine hydroxylase positive. Color image is available online.

The difference in endothelium-mediated vasodilation response between the two groups was likely mediated via endothelial TRPV 4, as the response to carbachol was abolished in both groups following treatment with the TRPV 4 channel antagonist HC067047 (Fig. 3A,B). This was further supported by immunohistochemical analysis, which demonstrated that endothelial TRPV 4 expression was 46% (SCI vs. CON: 0.5 ± 0.1 a.u. vs. 1 ± 0.1 a.u., respectively) lower in rats with SCI (Fig. 3G). Further, a 34% (SCI vs. CON: 0.7 ± 0.1 a.u. vs. 1 ± 0.1 a.u., respectively) lower expression of endothelial CD31 in the SCI is suggestive of loss of endothelial cells or changes in their function (Fig. 3G).

Morphological and structural changes in the MCA after SCI

Immunohistochemical analyses revealed there is a marked 50% (SCI vs. CON: 1.5 ± 0.2 a.u. vs. 1 ± 0.1 a.u., respectively) increase in COL 1 deposition in the media of the MCA after SCI (Fig. 4A). This is associated with a significant reduction in SMA staining. However, there was no significant difference in wall thickness, lumen diameter, or wall to lumen ratio (Fig. 4B). Perivascular sympathetic innervation of MCAs, as revealed by TH⁺ axonal density, was also not affected by SCI (Fig. 4C).

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FIG. 4.

Reduced baseline CBF and impaired spatial short-term memory are observed after prolonged high-thoracic SCI. (A) Representative MRI images and CBF heat maps from the controls (CON) and rats with SCI (SCI). Major brain regions associated with memory are labeled in T2 scan images (cortex in red, hippocampus in green, and basal forebrain in purple.) (B) Global CBF is reduced after chronic SCI, indicating lack of blood perfusion to the brain. (C) Brain regions associated with memory show reduced CBF after chronic SCI. (D) D-index of the NORT is significantly reduced in the SCI group. Injured animals exhibit significantly less of a preference for the novel object compared with controls. *Indicates significantly different from CON (p < 0.05); unpaired t test. Values are mean ± SEM. ASL, arterial spin labeling; CBF, cerebral blood flow; CON, uninjured animals; MRI, magnetic resonance imaging; NORT, novel object recognition test; SCI, T3-transected animals; SEM, standard error of the mean. Color image is available online.

Chronic SCI is associated with a reduction in CBF

Resting CBF was 32% (SCI vs. CON: 48.7 ± 3.5 mL/100 g/min vs. 71.7 ± 7.0 mL/100 g/min, respectively) lower in T3-SCI (Fig. 2A,B), indicating reduced brain perfusion following chronic SCI. Moreover, regional flow in SCI was significantly reduced in the hippocampus (32%, SCI vs. CON: 44.3 ± 4.5 vs. 65.0 ± 7.2 mL/100 g/min, respectively, Fig. 4C). No effects were seen on CBF in the basal forebrain or cortex.

SCI disrupts short-term memory

The control rats showed a higher preference for the novel object (D index = 81.1 ± 13.9%). In contrast, the rats with chronic SCI had significantly reduced preference for the novel object (D index = 62.6 ± 19.2%), suggesting impairment in short-term retention memory (Fig. 2D).

Future perspectives

We present an experimental high-thoracic SCI model that leads to endothelial dysfunction, structural remodeling of the cerebral arteries, global reduction in CBF, and vascular-cognitive impairment, which has now been identified as a serious disease burden in individuals with SCI. ^5,7,15,41 Interestingly, there appears to be a therapeutic time window between the establishment of vascular fibrosis and the onset of cognitive symptoms, offering various directions for upcoming research. Future studies could explore a number of intriguing avenues, such as spatiotemporal changes in expression of specific receptor subtypes to facilitate testing of potential drugs and other interventions for early prevention of vascular-cognitive impairment following SCI. For instance, upregulation of TRPV 4 channel activity may be a viable therapy for mitigating loss of cerebrovascular function. JNc-440, a designed molecule to strengthen the interaction between TRPV 4 and activated potassium channel 3 (KCa2.3), has been recently reported to enhance endothelial-induced vasodilation and exert anti-hypertensive effects. ⁴² A possible clinically relevant intervention could also be physical exercise, which has been shown to have significant benefits on endothelial function. ^43,44 Moreover, a better understanding of SCI-induced structural and functional changes in cerebral microvessels is needed. Alterations in the endothelial health of microvessels will likely provide deeper insight into the underlying mechanisms of vascular-cognitive impairment after chronic SCI and provide novel therapeutic targets. ⁴⁵