Mesenteric vascular dysregulation and intestinal inflammation accompanies experimental spinal cord injury

Abstract

Cervical and high thoracic spinal cord injury (SCI) drastically impairs autonomic nervous system function. Individuals with SCI at thoracic spinal level 5 (T5) or higher often present cardiovascular disorders that include resting systemic arterial hypotension. Gastrointestinal (GI) tissues are critically dependent upon adequate blood flow and even brief periods of visceral hypoxia triggers GI dysmotility. The aim of this study was to test the hypothesis that T3-SCI induces visceral hypoperfusion, diminished postprandial vascular reflexes, and concomitant visceral inflammation. We measured in vivo systemic arterial blood pressure and superior mesenteric artery (SMA) and duodenal blood flow in anesthetized T3-SCI rats at 3 days and 3 wk postinjury either fasted or following enteral feeding of a liquid mixed-nutrient meal (Ensure). In separate cohorts of fasted T3-SCI rats, markers of intestinal inflammation were assayed by qRT-PCR. Our results show that T3-SCI rats displayed significantly reduced SMA blood flow under all experimental conditions (P < 0.05). Specifically, the anticipated elevation of SMA blood flow in response to duodenal nutrient infusion (postprandial hyperemia) was either delayed or absent after T3-SCI. The dysregulated SMA blood flow in acutely injured T3-SCI rats coincides with abnormal intestinal morphology and elevation of inflammatory markers, all of which resolve after 3 wk. Specifically, Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) were acutely elevated following T3-SCI. Our data suggest that arterial hypotension diminishes mesenteric blood flow necessary to meet mucosal demands at rest and during digestion. The resulting GI ischemia and low-grade inflammation may be an underlying pathology leading to GI dysfunction seen following acute T3-SCI.

in addition to the catastrophic sensory and motor losses following spinal cord injury (SCI), autonomic nervous system dysfunction is also widely recognized (30). Furthermore, gastrointestinal (GI) dysmotility is observed clinically immediately after SCI (28, 71) and may persist for years after the initial injury (3, 12, 14, 37, 53, 56, 70). Dysfunction of the digestive organs following experimental SCI includes reduced gastric motility and gastric emptying, abnormal response to GI peptides, and reduced nutrient absorption. Each of these comorbidities contributes to diminished long-term quality of life after SCI (43).

The principal functions of the GI tract, the digestion and absorption of nutrients and the maintenance of proper fluid balance, require adequate blood flow to GI tissues. The primary vascular perfusion occurs through the splanchnic vascular bed that consists of the celiac, superior mesenteric, and inferior mesenteric arteries (35). The distal esophagus, stomach, and the proximal duodenum are vascularized by the celiac trunk which supplies three main branches: the left gastric artery, the common hepatic artery, and the splenic artery. The left and right gastric arteries are responsible for the lesser curvature, whereas the left gastroepiploic and right gastroepiploic arteries feed the greater curvature. The duodenum has a “dual” blood supply, arising from both the celiac trunk and the superior mesenteric artery (SMA). This vascular arrangement reflects the importance of blood supply, and the GI tract is one of the most highly perfused organ systems in the body whereby resting GI blood flow can reach ~20–25% of the total cardiac output (10).

Postprandial hyperemia, the global increase in blood flow to the GI tract following a meal, is a critical reflex for adequate GI function and has been demonstrated to result from the exposure of the intestinal mucosa to nutrients in concert with the release of GI peptides (11). The postprandial reflex involves a concurrent increase in blood flow through both the celiac and superior mesenteric arteries (58). Multiple mechanisms responsible for postprandial hyperemia have been proposed including local presynaptic activation of vasodilation by nitric oxide release (48), vago-vagal reflex activation (33), and inhibition of medullary presympathetic vasomotor neurons by vagal afferent input (50).

Individuals with spinal cord lesions, particularly those rostral to T5, present with diminished sympathetic tone due to disruption of the descending fibers of the medullary presympathetic vasomotor neurons. Loss of these presympathetic vasomotor neurons provokes cardiovascular instability, arterial hypotension, and pooling of blood in the extremities that has been documented clinically (68) and experimentally (31). Vascular hypotension and pooling of blood in the extremities may predispose the GI tract to hypoperfusion following SCI.

Reduced GI blood flow over an extended period of time deprives GI tissues of the oxygen needed to maintain organ integrity (11). The resulting ischemia and restoration of adequate blood flow provokes a multifactorial tissue injury response including 1) intercellular adhesion molecule-1 (Icam1) mediated increase in adherent leukocytes; 2) upregulation of chemokines, particularly monocyte chemotactic protein (Ccl2); 3) macrophage activation by macrophage inflammatory protein-1α (Ccl3); and 4) proinflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)1β, and IL6 (19, 67).

In the present work, we employed our established rodent model of T3 spinal level SCI to investigate 1) if T3-SCI leads to reduced mean arterial blood pressure (BP) and reduced resting blood flow within the superior mesenteric artery supplying the mesenteric bed; 2) if T3-SCI diminishes postprandial vascular reflexes; 3) if local duodenal tissue perfusion increases in response to nutrient infusion; and 4) if T3-SCI provokes concomitant histopathologic changes and inflammation of the small intestine.

METHODS

All procedures were performed following National Institutes of Health guidelines and under the approval of the Institutional Animal Care and Use Committee at the Penn State University College of Medicine.

Animals

Male Wistar rats (Hsd:WI, Stock 001, Harlan, Indianapolis, IN) ≥8 wk of age, initially weighing 175–200 g, were used for all experimental procedures. Rats (n = 116) were housed in a temperature-controlled room (23°C) on a 12:12-h light-dark cycle with unlimited access to food and water. After surgical manipulation was completed, rats were housed singly and observed twice a day. Each rat was randomly assigned to one of two surgical manipulations; surgical controls (in which the T3 spinal cord was exposed by laminectomy) or T3-SCI. At the same time, animals were also randomly assigned to one of two postsurgical survival times.

Surgical Procedures and Animal Care

Animals were anesthetized with a mixture of 3–5% isoflurane in oxygen (400–600 ml/min), and surgery for T3-SCI using the Infinite Horizons device was performed using established aseptic surgical techniques. When the rat was no longer responsive to toe pinch or palpebral reflex, the surgical site overlying the vertebrae from the interscapular region to midthoracic region was shaved and cleaned with three alternating scrubs of chlorhexidine and alcohol. Animals were maintained at 35.5–37.5°C on a feedback-controlled heating block, and rectal temperature was monitored continuously. The location of the elongated T1 and T2 spinous processes were determined by midline palpation. A 3- to 5-cm midline incision of the skin overlying the T1–T3 vertebrae was performed and the muscle attachments to the T1–T3 vertebrae were cleared by blunt dissection, taking care not to damage the vascular supply to the dorsal nuchal adipose tissue. Using fine-tipped rongeurs, the spinous process and the laminae of the T2 vertebra were removed laterally to the superior articular processes.

Rats receiving T3-SCI (n = 61) were transferred to the Infinite Horizons spinal contusion injury device (Precision Systems and Instrumentation, Fairfax, VA). The adjacent T1 and T3 vertebrae were secured into the device and the torso of the animal was suspended slightly above the platform. After the exposed spinal cord was centered beneath the impactor tip, a 300-kdyn impact (15-s dwell time) was initiated. This level of injury produces a consistent and reliable neurological and histological outcome whereby animals exhibit a residual, chronic, locomotor deficit and severe loss of the spinal cord white matter. After removal from the contusion device, all surgical incisions were closed in reverse anatomical order with absorbable suture (Vicryl 4–0) for internal sutures and skin closure with wound clips. Wound clips were removed 5–7 days following surgery. Surgical controls (n = 55) underwent all procedures except for the contusion injury. A total of 8 T3-SCI rats were lost from the study. Two rats died from unspecified surgical complications (one destined for 3 day in vivo physiology, one destined for 3 wk tissue harvest) and six rats (all utilized for 3 day in vivo physiology) were removed following post hoc verification of inadequate lesion severity.

Postoperatively, rats were administered supplemental fluids by subcutaneous injection of 5 ml warmed lactated Ringer’s solution and stabilized in an incubator (37°C) until fully recovered from anesthesia. Afterward, animals were monitored daily for any signs of infection or complications from surgery. Rats received extended-release analgesics (buprenorphine SR, 1 mg/kg ip, Pfizer Animal Health, Lititz, PA) at time of surgery, then antibiotics (enrofloxacin, 2.5 mg/kg) and subcutaneous supplemental fluids (5–10 ml lactated Ringer) twice daily for 5 days after surgery. Because of the reduction in locomotor capacity after T3-SCI, a reservoir of chow was placed at head level to facilitate ease of access for feeding. All T3-SCI rats ingested a measureable amount each day, thereby confirming that access to chow was available. Body weights and food weights were recorded each morning. T3-SCI rats received bladder expression and ventrum inspection twice daily until the return of spontaneous voiding occurred.

In Vivo Physiological Instrumentation

After 3 days (n = 17 T3-SCI, n = 18 control) or 3 wk (n = 5 T3-SCI, n = 5 control) following the initial surgery, animals were fasted overnight, water provided ad libitum, before being deeply reanesthetized with isoflurane (3–5%, 400–600 ml/min flow rate) for in vivo physiological instrumentation. Animals were placed on a feedback-controlled warming pad (TCAT 2LV, Physitemp Instruments, Clifton, NJ) and maintained at 37 ± 1°C for the duration of the experiment.

Tracheal cannulation.

Once fully anesthetized for physiological instrumentation, the animal was tracheally intubated by way of a 1- to 2-cm midline incision on the ventral side of the neck caudal to the mandible toward the sternal notch. The underlying strap muscles were separated using blunt dissection at the midline to expose the trachea. The exposed trachea was isolated from the underlying esophagus to place a loop of 3-0 Ethilon suture between the trachea and esophagus to form a ligature. The trachea was opened ventrally by making a small cut in the membrane between two of the cartilaginous rings of the trachea just inferior to the thyroid gland. A small piece of polyethylene tubing (PE-270, 5 mm in length and beveled at one end) was inserted into the trachea and secured in place with the ligature. The strap muscles were returned to their proper anatomic location and the overlying skin was secured around the tracheal tube with 3-0 ethilon. Tracheal intubation maintains an open airway and facilitates clearing of respiratory secretions if necessary.

Femoral arterial and venous catheterization.

After intubation was completed, the femoral artery and adjacent vein or tributaries were exposed, within the region of the femoral triangle, via a small skin incision at the intersection of the inguinum and proximal thigh. Connective tissue was cleared from the femoral artery and vein proximally to the inguinal ligament. The proximal and distal extremes of the exposed artery were gently ligated with 4-0 silk suture. To monitor arterial blood pressure, the femoral artery was hemisected and a sterile PE-50 catheter was inserted in the direction of the abdominal aorta toward the heart. To avoid disrupting the arterial endothelium, thus potentially confounding arterial pressure readings, the femoral catheter pressure was advanced so that it terminated in the larger-diameter common iliac or in the descending aorta where chance of disrupting the endothelium is reduced. The proximal end of the artery and catheter were fully secured with the 4-0 silk suture and exteriorized. The wound margin was closed with wound clips.

Transonic flow probe.

Animals were simultaneously weaned off isoflurane inhalation and deeply anesthetized with thiobutabarbital (Inactin; Sigma, St. Louis, MO; 75–150 mg/kg iv), which does not affect long-term cardiovascular (7) or gastrointestinal (45) autonomic function. The rate of Inactin infusion was monitored in conjunction with a resulting momentary drop in arterial BP that quickly returns to normal (SYS-BP1, World Precision Instruments, Sarasota, FL). Once a deep state of anesthesia was achieved, a midline laparotomy was made and the intestines were gently displaced laterally to allow the exposure of the abdominal aorta at the level of the left renal artery. The SMA was carefully cleared of connective tissue immediately distal to where it passed over the caudal vena cava to allow for the perivascular flow probe (1PR, Transonic Systems, Ithaca, NY) to be positioned alongside the artery so as not to restrict blood flow.

In animals that were to receive duodenal infusion of a liquid mixed-nutrient mean (Ensure), a PE-90 catheter was inserted into the proximal duodenum through a small incision in the stomach adjacent to the pylorus and secured with a purse-string suture before positioning the perivascular flow probe around the SMA. The Ensure was delivered through the catheter by way of a syringe driven by a syringe pump (Razel R99-E, Fisher Scientific) set at an infusion rate of 1 ml/h.

Laser-Doppler flow probe.

After placement of the Transonic flow probe and duodenal catheter the retracted viscera were returned to the proper anatomic location. In a subset of the 3-day animals that were simultaneously implanted with the Transonic flow probe (n = 8 T3-SCI, n = 6 control) and for all 3-wk animals that were implanted with the Transonic flow probe (n = 5 T3-SCI, n = 5 control as enumerated above), a laser-Doppler flow probe (BLF22, Transonic Systems) was positioned in close contact with the mesenteric border of the duodenum immediately distal to the region where the tip of the implanted catheter terminated. Once a stable reading was achieved from the flow probe, the incision was closed around the implanted flow probe and the skin loosely secured with stainless steel wound clips. Animals were allowed to stabilize for 1 h before data collection was initiated.

Blood Flow Analysis

At the initiation of the stabilization period, the femoral arterial catheter was attached to a pressure transducer (BP-1, World Precision Instruments). Data from the flowmeter (T206, Transonic Systems), blood pressure monitor, and laser-Doppler flow probe were continuously recorded to computer (Spike 2, Cambridge Electronic Design, Cambridge, UK). Flow probe signals were filtered at 0.1–10 Hz and converted to blood flow in milliliters per minute and normalized for body weight. The mean percent change in Doppler output from baseline was calculated for each experimental manipulation. The effect of duodenal infusion was compared with the average blood flow rate of the 10 min preceding the infusion. Peak flow rate was calculated as the highest achieved value during the 1 h following the infusion.

Tissue Harvest

Gastrointestinal tissue.

Rats were deeply anesthetized with isoflurane until nonresponsive to toe pinch. Quickly, the rats were decapitated and the abdomen was opened via a midline incision. GI tissue (stomach and proximal duodenum) was taken at 1, 3, 7 days, or 3 wk following T3-SCI or post control surgery (n = 8 per group with one 3-wk SCI mortality as noted above). Following GI tissue isolation, a small tissue sample from both the stomach and duodenum, each weighing ~200 mg, was removed and placed in aluminum foil and immediately frozen in liquid nitrogen then transferred to a −80°C freezer until used for qRT-PCR. In the same animals, an adjacent section of GI tissue was removed (as above) and placed in room temperature 10% neutral buffered formalin (NBF) for histological processing.

At the conclusion of in vivo physiological experiments, deeply anesthetized rats were transcardially perfused with heparinized phosphate-buffered saline (PBS) until fully exsanguinated and followed immediately with PBS containing 4% paraformaldehyde. The spinal cord encompassing the lesion level was removed and refrigerated overnight in PBS containing 20% sucrose and 4% paraformaldehyde.

Histological Processing

Intestine.

Formalin-fixed tissue from the duodenum 1 cm distal to the pylorus was processed in an automated Tissue-Tek VIP processor and paraffin-embedded with a Tissue-Tek TEC embedding station (Sakura Finetek USA, Torrance, CA). Sections were cut at 6 µm for routine hematoxylin and eosin (H&E) staining.

Intestinal sections were examined by an American College of Veterinary Pathologists diplomate blinded to treatment (T. K. Cooper). All images were obtained with an Olympus BX51 microscope and DP71 digital camera using cellSens Standard 1.6 imaging software (Olympus America, Center Valley, PA).

Multiple (3–6) random tissue sections were quantified as described previously (20) and the following measures determined: 1) villus height and width; 2) crypt depth and width; and 3) villus:crypt height ratio was calculated. In each case, 10 independent measurements for each variable were collected from at least 3 different intestinal sections. Semiquantitative measurements of inflammation scoring were made on a modified scale (Table 1; adapted from Refs. 2 and 4).

Table 1.

Semiquantitative measurements of inflammation scoring for gastrointestinal tissue

Grade	Description
Grade 0	No change from normal tissue
Grade 1	One or a few multifocal mononuclear cell infiltrates in the lamina propria
Grade 2	Lesions involve more of the intestine than grade 1 lesions, and/or are more frequent. Typical changes include several multifocal, mild inflammatory cell infiltrates in the lamina propria composed primarily of mononuclear cells with a few neutrophils. Inflammation rarely involves the submucosa.
Grade 3	Lesions involve a large area of the mucosa or are more frequent than grade 2 lesions. Inflammation is moderate and involves the submucosa but is not transmural. Inflammatory cells are a mixture of mononuclear cells as well as neutrophils, and crypt abscesses are sometimes observed. Small epithelial erosions are occasionally present.
Grade 4	Lesions involve most of the intestinal section and are more severe than grade 3 lesions. Inflammation is severe, including mononuclear cells and neutrophils, and can be transmural. Crypt abscesses and ulcers are present.

Spinal cord lesion center.

For histological staining of T3-SCI lesion extent, tissue was sectioned (40-μm thick), and alternating sections were mounted on gelatin-coated slides. To compare lesion severity with the spinal cords of control animals, spinal cord sections were stained with luxol fast blue (LFB) to visualize myelinated fibers. LFB-stained slides were digitally imaged on a Zeiss Axioscope light microscope and Axiocam CCD camera, imported into Adobe Photoshop, and contrast digitally adjusted to allow consistent identification of LFB-stained (i.e., spared) white matter. For individual images, the boundaries of the tissue slice were outlined to determine cross-sectional area. A separate threshold histogram was generated and the pixels corresponding to LFB staining above background were selected. These pixels were quantified and expressed per unit cross-sectional area (38). The lesion epicenter was defined as the section with the least proportion of LFB-stained tissue. The proximity of the T3 lesion center to the cervical enlargement precluded an appropriate determination of spinal cord cross-sectional area in undamaged tissue rostral to the injury (i.e., damaged tissue extended into the cervical enlargement as described in 60). Therefore, it was necessary that the cross-sectional area of the intact spinal cords at T3 of comparably sized animals be determined for normalization purposes. LFB-stained myelin in injured tissue was then expressed as a percentage of the total spinal cord cross-sectional area as would be predicted by the intact tissue.

On the basis of previous reports (52, 61, 62) we determined a priori that animals euthanized 3 days following surgery in which LFB staining at the lesion epicenter accounted for ≤ 25% of the region occupied by white matter would be categorized as severe spinal injury; those with ≥ 25% LFB staining were excluded from further analysis (n = 6 T3-SCI rats met this criterion). This criterion is based upon the observation that considerable LFB-staining remains within the lesion center in the 1–3 days following injury, although the majority of the LFB-stained tissue likely consists of remaining myelinated axons as well as myelin debris in a loose fibrous matrix as reported previously (62). Historically, our animals with the same 300-kdyn injury that are euthanized 3 wk after injury display ≤ 5% of LFB staining above threshold as the lesion center is clear of cellular debris. After 3 wk any remaining LFB staining is usually confined to a thin band within the ventrolateral white matter in a manner consistent with previous reports characterizing a 200-kdyn injury level (52).

RNA Isolation, Reverse Transcription Reaction, and qRT-PCR

Quantitative reverse transcriptase PCR (qRT-PCR) was used to quantify the level of inflammatory mediators present at the assigned time points. Tissue sections from the cranial gastric corpus and proximal duodenum were analyzed for intercellular adhesion molecule-1 (Icam1), monocyte chemotactic protein (Ccl2), and macrophage inflammatory protein-1α (Ccl3), following T3-SCI and control surgery. Nomenclature is presented according to Rat Genome Nomenclature Committee guidelines (http://www.informatics.jax.org/mgihome/nomen/gene.shtml) along with more common, informal, usage. These particular molecules are commonly reported in the scientific literature and were selected as reliable biomarkers of gastrointestinal pathophysiology (see 19).

Whole GI tissue sections were used for RNA isolation. A small section of GI tissue weighing 50–100 mg was cut away from the whole tissue section and used for RNA isolation. RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA) and RNeasy Microkit procedures (Qiagen, Valencia, CA). Briefly, frozen tissue was homogenized in TRIzol using a glass homogenizer and Teflon pestle on ice, chloroform was added to lysate, and the mixture was centrifuged in microcentrifuge tubes to separate RNA. Ethanol was added to the upper aqueous phase, and the mixture was applied to an RNeasy spin column and filtered by centrifugation. After several washes, the samples were subjected to an elution step using RNase-free water. Reverse transcription (RT) was conducted using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). For RT, ~1 μg of RNA from each sample was added to random primers (10 × ), dNTP (25 × ), MultiScribe reverse transcriptase (50 U/μl), RT buffer (10 × ), and RNase Inhibitor (20 U/μl) and incubated in a thermal cycler (Techne TC-412, Barloworld Scientific, Burlington, NJ) for 10 min at 25°C, then for 120 min at 37°C. Primers for Actb (β-actin) were a QuantiTect Primer Assay (Rn_Actb_1_SG QuantiTect Primer Assay QT00193473, Qiagen, Frederick, MD). Primers for Icam1, Ccl2 (MCP-1) and Ccl3 (MIP-1α) were designed using Primer Express (Applied Biosystems). The forward and reverse primer pairs used for these studies are shown in Table 2.

Table 2.

Forward and reverse primer sequences for quantitative real-time PCR (qRT-PCR)

Gene	Forward Primer	Reverse Primer
Ccl2 (MCP-1)	5′-TCTCTGTCACGCTTCTGGGCCT-3′	5′-TAGCAGCAGGTGAGTGGGGCA-3′
Ccl3 (MIP-1α)	5′-TGACACCCCGACTGCCTGCT-3′	5′-TGACACCCGGCTGGGACCAA-3′
Icam1	5′-TGCCAGCCCGGAGGATCACA-3′	5′-CGGGAGCTAAAGGCACGGCA-3′

For real-time PCR, SYBR Green 2 × Master Mix (Qiagen), forward and reverse primers (100 μM), and RT product (1 μl of a 1:16 dilution) were added to a 384-well plate. The cycling parameters consisted of an initial 2-min incubation at 50°C, followed by 10 min at 95°C, then 15 s at 95°C, a 30-s annealing step at 55°C, and a 30-s extension step at 72°C (55 cycles). A dissociation step (15 s at 95°C) was added following 55 cycles to determine specificity of primers. In this assay, the dissociation step confirmed the absence of nonspecific amplifications. Quantity of Icam1, Ccl2 (MCP-1), and Ccl3 (MIP-1α) mRNA was based on a standard curve and normalized to Actb (β-actin) mRNA (ABI QuantStudio 12KFlex with available OpenArray block, Applied Biosystems). The suitability of Actb as an internal control was assessed through analysis of the raw data between groups, and no variability of Actb was detected.

Statistical Analysis

Results are expressed as means ± SE with significance defined as P < 0.05. Body weight and MEI measurements from 3-day survival rats did not significantly differ from rats destined to survive 3 wk. Therefore, these 3-day measures were collapsed for the within-groups two-way ANOVA comparison followed by Tukey post hoc analysis. Between-groups results from in vivo blood flow studies were compared by one-way ANOVA and Tukey post hoc analysis or paired t-test as appropriate. Group results from qRT-PCR were compared by between-groups two-way ANOVA and Tukey post hoc analysis or paired t-test as appropriate. Statistical analysis was performed using SigmaPlot for Windows (SPSS, Chicago, IL).

RESULTS

Assessment of T3-SCI Histological Severity, Reduction of Spontaneous Feeding, and Loss of Body Weight

The severity of experimental T3-SCI was verified on the basis of the reduction of LFB-stained white matter at the T3 spinal cord segment (Fig. 1A). The percent area of white matter at the lesion epicenter of 3-day T3-SCI rats was significantly reduced in comparison to T3-control animals (Fig. 1B; P < 0.05). At 3 wk, when the postinjury progression of the lesion epicenter has relatively stabilized and the lesion boundaries are more clearly defined (22), the percent area of white matter at the lesion epicenter of 3-wk T3-SCI rats was significantly reduced in comparison to age-matched T3-control animals (Fig. 1B; P < 0.05). The data for control animals were pooled in Fig. 1B for clarity. These data are comparable to the injury extent reported previously and indicate the severity of our injury model (44, 57, 60, 61).

Fig. 1.

A: Luxol-stained white matter from T3 spinal cords of control (left), 3-day postoperative (middle), and 3-wk postoperative (right) rats (scale bar = 1 mm). B: graphic summary of the percent sparing of white matter at the lesion epicenter of control, 3-day, or 3-wk rats following a 300-kdyn contusion spinal cord injury (SCI) (*P < 0.05 vs. age-matched controls; †P < 0.05 vs. 3-day T3-SCI).

At 3 days following surgery, the change in body weight between T3-SCI and control animals was −22.5 ± 2.0 vs. 1.2 ± 1.5 g, respectively. When normalized as percentage of preoperative weight, T3-SCI rats displayed significantly greater weight loss than surgical controls for the comparable time period across the duration of the study (Fig. 2A; P < 0.05).

Fig. 2.

Postoperative body weight and food intake are significantly lower in T3-SCI animals. Compared with age-matched control animals, the postoperative body weight (expressed as percentage of preoperative weight) is significantly lower following T3-SCI for the duration of the experiment (A). The mean energy intake is significantly reduced following T3-SCI for the first 2 wk compared with their age-matched cohort (B). For all measures *P < 0.05 vs. age-matched control.

Regardless of ease of physical access to chow, spontaneous feeding is suppressed following T3-SCI when gastric motility is compromised. When normalized as the mean energy intake (MEI; defined as kcal·day⁻¹·100 g body wt⁻¹) the spontaneous feeding for T3-SCI animals in the present study was significantly lower than controls for every comparable time point until the third week of the study (Fig. 2B; P < 0.05).

As demonstrated in our previous studies (43, 44, 60), T3-SCI reduced the area of intact white matter, body weight, and caloric intake. These data further verify the profound severity, effectiveness, and reproducibility of our surgical procedures for T3-SCI and surgical control animals. On the basis of these criteria, all animals in these groups were selected for further data analysis.

Basal Mean Arterial Blood Pressure and Mesenteric Blood Flow Are Decreased in T3-SCI Rats

Prior to the initiation of duodenal nutrient infusion, the baseline systemic mean arterial pressure (MAP) of Inactin-anesthetized T3-SCI rats was significantly lower than the MAP of age-matched surgical control animals (Table 3, baseline; P < 0.05). Following normalization for body weight, basal SMA blood flow in fasted 3-day T3-SCI rats was significantly lower than controls (2.2 ± 0.2 vs. 3.4 ± 0.4 ml·min⁻¹·100 g body wt⁻¹, respectively; P < 0.05). In the age-matched cohort of animals tested at 3 wk after surgery, normalized basal SMA flow was significantly lower in T3-SCI rats compared with controls (1.2 ± 0.2 vs. 2.1 ± 0.2 ml·min⁻¹·100 g body wt⁻¹, respectively; P < 0.05). These results confirm that T3-SCI in the rat produces arterial vascular hypotension and hypoperfusion of the splanchnic vascular beds.

Table 3.

Mean arterial pressure is not altered by Ensure infusion in 3-day T3-SCI, 3-day surgical controls, 3-wk T3-SCI, and 3-wk controls

	Baseline	30-min Infusion	60-min Infusion
3-day Control	112.9 ± 4.3	109.2 ± 2.4	105.2 ± 5.0
3-day T3-SCI	74.8 ± 4.9*	69.9 ± 5.7*	65.9 ± 7.0*
3-wk Control	127.5 ± 6.1	122.0 ± 4.5	112.8 ± 3.2
3-wk T3-SCI	90.2 ± 3.4†	90.8 ± 4.2†	88.6 ± 3.2†

Values are in mmHg and are presented as means ± SE.

^*P < 0.05 vs. 3-day control.

^†P < 0.05 vs. 3-wk control.

Postprandial Mesenteric Arterial Reflexes Are Reduced in T3-SCI Rats

Following duodenal infusion of a liquid mixed-nutrient meal (Ensure, delivered at 1 ml/h), T3-SCI rats fail to exhibit the increase in SMA blood flow that is demonstrated by control animals (Fig. 3A). During 30 and 60 min infusion of Ensure into the duodenum, the MAP remained significantly different between T3-SCI and control rats (Table 3; P < 0.05); however, in both T3-SCI and control rats, duodenal infusion of Ensure did not significantly change MAP from preinfusion baseline values (Table 3; P > 0.05).

Fig. 3.

Postprandial hyperemia is significantly lower in T3-SCI animals. Representative traces (A) illustrating the normal postprandial hyperemia from a 3-day control rat (top trace), whereas postprandial SMA blood flow from 3-day T3-SCI rats (second trace) did not demonstrate a response to duodenal perfusion of a mixed-nutrient meal (Ensure; infusion rate was 1 ml/h). This disruption of postprandial response continued through 3 wk following T3-SCI. Arrows depict the initiation of Ensure administration for each representative subject. B: the peak volume of SMA blood flow reached during the intraduodenal infusion period was also significantly reduced in 3-day and 3-wk T3-SCI rats. C: local tissue perfusion was measured by laser-Doppler flow of the duodenal serosa. Compared with controls, the percent change in Doppler signal vs. baseline flow was significantly lower only in 3-day T3-SCI rats. Values expressed as means ± SE; *P < 0.05 vs. control.

During the 60 min intraduodenal infusion of Ensure, the peak blood flow within the SMA was significantly lower in 3-day T3-SCI rats following the nutrient challenge (Fig. 3B; P < 0.05) compared with controls. This significant difference in peak blood flow persisted in the rats tested 3 wk after surgery (1.4 ± 0.2 vs. 2.7 ± 0.2 ml·min⁻¹·100 g body wt⁻¹; P < 0.05). Laser-Doppler analysis of the local duodenal perfusion in the region of Ensure infusion demonstrated that the percent change in blood flow within the duodenal serosa of surgical control rats was significantly elevated from baseline in comparison to 3-day T3-SCI rats which did not increase during enteral feeding (Fig. 3C; P < 0.05). At 3 wk after surgery, there was no significant difference in serosal duodenal blood flow between control and T3-SCI rats (127 ± 16% vs. 124 ± 17%, respectively; P > 0.05).

These results indicate that T3-SCI in the rat diminishes the mesenteric vascular reflexes in response to feeding. Local enterically-mediated changes in duodenal blood flow are also diminished in these same animals during the acute (3 day) phase of injury, but local regulation of duodenal microcirculation returns in longer-term survival periods.

T3-SCI Provokes Gastrointestinal Tissue Necrosis and Shortening of Mucosal Villi

Upon removal of the GI tract from 3-day T3-SCI animals, it was qualitatively observed that GI mucosal tissue was compromised 3 days following injury compared with controls, whereas mucosal integrity after 3 wk was unremarkable (Fig. 4). For example, sections of the GI tract were described as atrophic, with hemoccult positive contents, and excised tissue was friable (data not shown). Extreme cases also included profound reduction in duodenal integrity, and regions of the small intestine revealed necrosis of mucosa and submucosa, neutrophil and macrophage infiltration, and fibroplasia of serosa and submucosa. The duodenum at 3 days following T3-SCI revealed a significant reduction in average mucosal villous height and width (Table 4, P < 0.05). The inflammatory score of randomly analyzed tissue segments was significantly elevated in the duodenum of 3-day T3-SCI rats (Table 4, P < 0.05), but not significantly different in 3-wk T3-SCI rats (Table 4, P > 0.05).

Fig. 4.

Representative images of H&E-stained duodenal sections after T3-SCI or control surgery. T3-SCI provokes altered mucosal architecture as evidenced by blunting of intestinal villi at 3 days following T3-SCI compared with surgical control animals. After 3 wk, the height and width of intestinal villi were similar for both T3-SCI and control animals. (×100, scale bar, 200 μm).

Table 4.

T3-SCI provokes an inflammatory response and blunting of mucosal villi in duodenal tissue at 3 days after injury

	Experimental Groups
	Control	T3-SCI
3 day
Average inflammatory score	0.4 ± 0.2	0.9 ± 0.1*
Average villus height, µm	435 ± 24	341 ± 11*
Average villus width, µm	122 ± 4	102 ± 2*
Average crypt depth, µm	149 ± 6	147 ± 10
Average crypt width, µm	52 ± 2	52 ± 2
Villus:crypt ratio	3 ± 0.1	2 ± 0.2
Villus height:width ratio	4 ± 0.2	3 ± 0.2
3 wk
Average inflammatory score	1.75 ± 0.9	1.75 ± 0.1
Average villus height, µm	513 ± 12	491 ± 23
Average villus width, µm	121 ± 4	113 ± 4
Average crypt depth, µm	178 ± 8	201 ± 8
Average crypt width, µm	48 ± 1	44 ± 1
Villus:crypt ratio	3 ± 0.1	2 ± 0.1
Villus height:width ratio	4 ± 0.2	4 ± 0.1

^*P < 0.05 vs. control.

These data demonstrate a continuum of impaired GI tissue health immediately following T3-SCI. Taken together, our anatomic and histological data verify the profound severity produced by our surgical procedures for T3-SCI compared with surgical control animals.

T3-SCI Increases Upper-GI Expression of Inflammatory Markers

To quantify upper-GI inflammation, total RNA was isolated to analyze expression of inflammatory markers commonly linked with GI inflammatory processes (19).

In our experimental T3-SCI conditions, gastric Ccl2 expression was not significantly different between T3-SCI and control (Fig. 5A; P > 0.05). Gastric expression of the chemokine Ccl3 demonstrated a significant increase (Fig. 5A; P < 0.05) at 1 day and 3 days following T3-SCI. However, after 1 wk, Ccl3 expression was not significantly different between T3-SCI and control (Fig. 5A; P > 0.05). The post-SCI expression of Icam1 demonstrated a significant increase at 1 day and 3 days following T3-SCI that returned to stable levels within 1 wk (Fig. 5B; P < 0.05).

Fig. 5.

Expression levels of gastric inflammatory markers mRNA after T3-SCI. A: gastric Ccl2 (MCP-1) mRNA expression was not significantly altered in T3-SCI rats. B: gastric Ccl3 (MIP-1α) mRNA expression demonstrated a significant (between-groups) elevation in T3-SCI rats at 1 day and 3 days compared with control animals matched for the same postoperative time point (denoted by lowercase a). C: gastric Icam1 mRNA expression was significantly elevated in T3-SCI rats at 1 day and 3 days compared with control animals matched for the same postoperative time point. Levels of Ccl3 and Icam1 returned to baseline by 1 wk postinjury. ^aP < 0.05, based on ANOVA, followed by Tukey post hoc test. Values expressed as means ± SE.

Consistent with our histology findings, duodenal Ccl2 expression was only significantly different between T3-SCI and control at 3 days after T3-SCI (Fig. 6A; P < 0.05). Duodenal expression of Ccl3 demonstrated a significant increase in T3-SCI rats at 1 day after injury (Fig. 6B; P < 0.05). The significant differences between T3-SCI rats are interpreted to reflect that Ccl3 returned to low levels beginning at 3 days onwards. The post-SCI expression of Icam1, however, demonstrated a significant increase at 1 day and 3 days following T3-SCI (Fig. 6C; P < 0.05) and returned to low levels by 1 wk following T3-SCI. Both control and T3-SCI rats had a significant increase in Icam1 levels over the 3 days postoperatively. The principal findings of these data indicate that animals with T3-SCI demonstrate a significant short-term GI inflammatory response immediately following injury.

Fig. 6.

Expression levels of duodenal inflammatory marker mRNA after T3-SCI. A: duodenal Ccl2 (MCP-1) mRNA expression demonstrated a significant elevation in T3-SCI rats only at the 3-day postoperative time point compared with control animals. B: the expression of duodenal Ccl3 (MIP-1α) mRNA demonstrated a significant elevation in T3-SCI rats only at 1 day postinjury compared with control animals matched for the same postoperative time point. Expression levels for T3-SCI returned to baseline by 3 days postoperative. C: duodenal Icam1 mRNA expression demonstrated a significant elevation in T3-SCI rats at 1 day postinjury and continuing through 3 days postinjury compared with control animals at the same postoperative time point. The peak response for T3-SCI rats occurred in 3-day survival rats. Levels of Ccl2, Ccl3, and Icam1 returned to baseline within 1 wk postinjury. ^aP < 0.05, based on ANOVA, followed by Tukey post hoc test. Values expressed as means ± SE.

DISCUSSION

The present experiments demonstrate that systemic cardiovascular derangements at 3 days following a severe T3-SCI include reduced splanchnic vascular competence at rest and following duodenal infusion of a liquid mixed-nutrient meal designed to model clinical enteric supplementation. Specifically, these data indicate that 1) the anticipated reduction in baseline mean arterial pressure is accompanied by significantly reduced basal blood flow rate through the SMA in rats 3 days after T3-SCI; 2) mean arterial pressure remains at baseline levels in response to enteral administration of a liquid mixed-nutrient meal in both control and T3-SCI rats; 3) T3-SCI rats have a significantly reduced postprandial mesenteric response following a liquid mixed-nutrient meal; 4) T3-SCI induced a brief, but significant elevation in the gastric expression of inflammatory cytokine transcripts for Icam1 and Ccl3 (MIP-1α); and 5) duodenal expression of Icam1 was most profoundly elevated after T3-SCI. The level of tissue loss at the lesion epicenter, coupled with the observed reduction in feeding and weight loss, is consistent with our previous findings in severe T3-SCI rats that demonstrated gastroparesis and delayed gastric emptying (44, 57, 60). These data lead us to propose that the clinically recognized vascular reflex deficits in the SCI population may extend to the splanchnic vascular bed that irrigates the GI tissues as demonstrated in our experimental model of high thoracic SCI, a though these deficits appear to be only during the early phase of injury. Furthermore, diminished splanchnic perfusion following T3-SCI may trigger the low-grade inflammation observed in GI tissues.

Systemic Vascular Compromise Following T3-SCI

A common consequence of SCI is systemic vascular dysfunction (42). Furthermore, human studies have shown that high-level (cervical) SCIs are accompanied by the most severe hypotension and bradycardia (15–17, 29). The sympathetic preganglionic neurons within the thoracic and lumbar spinal cord normally receive descending inputs, including that from the medullary cardiovascular centers. Interruption of these supraspinal fibers following SCI results in low resting systemic arterial BP, loss of ability to regulate arterial BP, low cardiac output, low venous return, and disturbed reflex control (30, 42). Our acute studies are in agreement with previous observations of a profound reduction in systemic arterial BP after experimental SCI (31, 69). Our observation that arterial BP only partially recovers after 3 wk is also in agreement with recent temporal studies demonstrating that arterial BP remains chronically suppressed following midthoracic spinal transection (69). Beyond the means by which injury was induced, several notable differences exist between our data and the above-mentioned study. The reduction in femoral arterial BP was qualitatively greater in our model than the aortic BP described from the previous report (69). Although our studies were in thiobutabarbital-anesthetized rats, rather than telemetrically implanted awake rats, this particular anesthetic has been reported to have no deleterious effects on cardiovascular function (7). Furthermore, our reduced femoral arterial BP during experimentation was similar to ranges previously reported in chronic SCI rats by Laird and colleagues (31). Therefore, we conclude that our observations are consistent with the post-SCI hypotension reported in the literature.

Visceral Hemodynamics Following T3-SCI

It is estimated that upwards of 70% of blood volume resides within the venous circulation. Vascular stasis coupled with the absence of lower-extremity muscle pumps and elevated venous flow resistance leads to venous pooling within extremities. The latter phenomena have been previously reported in experimental models of SCI and may contribute to mesenteric insufficiency (31). Our data demonstrated a reduction in basal blood flow within the SMA. The principal blood supply to the stomach and intestines arises from the gastric branch of the celiac trunk and superior and inferior mesenteric arteries and are collectively referred to as the splanchnic vascular bed. Although only one vessel from this triad of splanchnic vessels was monitored, diminished perfusion throughout the splanchnic vascular bed was inferred for all T3-SCI rats.

Instances of chronic mesenteric hypoperfusion in atherosclerotic disease or acute mesenteric hypoperfusion following strenuous exercise often report the presentation of abdominal or intestinal angina and hemorrhage (54, 64). With regard to the elderly population, mesenteric stenosis occurs with increasing frequency over 65 yr of age (21). Symptomatic presentations were noted to occur during the postprandial phase and underscore the ramifications of widespread insufficiency of splanchnic circulation. Our findings provide initial evidence that the mesentery of SCI subjects may be vulnerable to the pathologies associated with ischemic events.

The postprandial dynamics of blood flow to the splanchnic organs in neurally intact animal models have been previously described (11, 58). Postprandial hyperemia in experimental animals subjects consists of a profound increase (ca. 200%) in regional GI blood flow in response to nutrients (35). This redistribution of blood flow is compensated by reflexive increase in cardiac output and a redistribution of flow from other tissues (11). In addition, there is substantial evidence that postprandial hyperemia is locally mediated within the intestinal microvasculature through a complex and not completely understood interplay of local oxygen titers, adenosine levels, prostaglandins, sodium-induced hyperosmolarity, and the degree of muscle deformation (39). Ultimately, these changes in microvasculature are under the influence of the hemodynamics of upstream mesenteric arteries. These larger-caliber supply arteries and arterioles are under greater influence from extrinsic sympathetic sources (reviewed in 23). One important mechanism in postprandial hyperemia involves the release of GI peptides that have been demonstrated to exert a role in regulating postprandial hemodynamic demand through a centrally mediated reflex (49, 50). Specifically, intestinal cholecystokinin (CCK) and gastric leptin activate subdiaphragmatic vagal afferents that, ultimately, terminate in the nucleus tractus solitarius (NTS). In addition to the role of the NTS toward the modulation of gastric-projecting preganglionic motoneurons in the vagal dorsal motor nucleus (DMN 46), CCK-sensitive afferents terminate upon a subpopulation of NTS neurons that directly project to select cardiovascular neurons in the rostral ventrolateral medulla (RVLM). Under normal conditions, activation of these RVLM neurons provokes an elevation of systemic sympathetic drive and vasoconstriction within skeletal muscle. Simultaneous input by NTS neurons that project to caudal ventrolateral medulla (CVLM) provokes a reduction in splanchnic sympathetic tone resulting in vasodilation within the mesentery (51). Presympathetic vasomotor projections from both the RVLM and CVLM descend through the spinal cord and are disrupted by T3-SCI. With particular emphasis on the rat, the segmental distribution of identified cardiovascular sympathetic preganglionic neurons begins principally at the second spinal thoracic segment and progresses caudally (18). Evidence from these experiments as well as that gathered from Doppler blood flow studies of the liver (65) suggests that visceral arterial blood flow is significantly diminished in rats with acute (24–76 h) SCI located at or above midthoracic (T5) spinal cord.

Inflammation in Visceral Organs Following T3-SCI

Although the GI mucosa is a richly perfused vascular bed in health, it is directly juxtaposed with the anaerobic and nonsterile lumen of the gut. As such, intestinal epithelial cells that line the mucosa experience a uniquely steep physiological oxygen gradient in comparison with other cells of the body. Thus the intestine is one of the most sensitive tissues to hypoxic insult and even brief periods of GI hypoxia induce the production of inflammatory mediators and dysmotility. Furthermore, there is evidence that hypoxia may be more deleterious to cells than complete anoxia (13). Experimental in vitro studies in which mitochondrial or glycolytic metabolism has been disrupted pharmacologically (thereby depleting ATP) have shown that minor reduction in ATP maintained for 12–24 h is sufficient to induce epithelial monolayer dysfunction (63). From a clinical standpoint, visceral hypoperfusion in the intensive-care patient leads to hypoxia and initiates an inflammatory cascade with consequent end-organ dysfunction, and cervical SCI patients are, indeed, susceptible to multiple organ dysfunction (55). On the basis of these observations, the dysregulation of mesenteric blood flow in acutely-injured T3 SCI rats suggests that arterial hypotension consequently diminishes mesenteric blood flow necessary to meet mucosal demands at rest and during digestion. We hypothesized that our observed GI hypoperfusion may be an underlying pathology leading to gastric dysfunction through the generalized mechanism of reduction in energy homeostasis and the initiation of cell damage, destruction, and death due to ischemia (40, 59). Furthermore, it is recognized that ischemia initiates an inflammatory cascade (73). However, caution must be exercised when extrapolating the data from ischemia-reperfusion models and our model of T3-SCI. The acute period of high-level SCI presents severe hypotension requiring vasopressor therapy (reviewed in 66). It is unclear, however, whether this period of so-called “neurogenic shock” produces a level, and duration, of mesenteric hypoperfusion that is comparable to the approximate 90% reduction of flow seen after SMA occlusion.

The reperfusion of ischemic tissues involves a known, biochemically mediated event involving the increased expression of adhesion molecules and chemokines (41). Beginning with early mast cell degranulation and histamine release (8, 27), the upregulation of adhesion molecules and chemokines forms the early line of defense in the intestinal mucosa and leads to an inflammatory pathway which promotes neurotoxicity, leukocyte (including lymphocytes, neutrophils, and monocytes), macrophage, and astrocyte recruitment (36), endothelial damage, hypoperfusion, and apoptosis (5, 59, 73). Utilizing our model of acute T3-SCI in rats, we demonstrated the effects of T3-SCI upon Icam1, Ccl2, and Ccl3 expression within the upper-GI tract which suggests the initiation of a low-grade inflammatory cascade following T3-SCI.

Implications of Gastrointestinal Vascular Dysregulation

It is generally recognized that the intestinal tract is acutely sensitive to traumatic events (1). The relationship of properly regulated GI blood flow with patient morbidity or mortality is well recognized in many instances of advanced aging, trauma, and critical illness (9, 34, 72). The implications of severely diminished blood flow to the GI tract following SCI are likely to mirror some aspects of these other clinical situations. Other models have shown that ischemic GI tissue reacts by releasing lactate as the mucosal-arterial Pco₂ gradient increases, indicating the initiation of anaerobic metabolism in the gut (26) and recruitment of proinflammatory cytokines and inflammatory markers. Therefore, if post-SCI hypoperfusion leads to ischemia, tissue damage and necrosis are likely to occur whereby the walls of the GI tract may become permeable, allowing bacteria to proliferate and translocate through the gut wall and into lymph nodes and blood vessels (6, 32). With inadequate splanchnic perfusion, multiple organ failure and death may ensue (25). The development of episodic hypertension, a phenomenon associated with massive sympathetic discharge that is triggered by noxious visceral or sensory stimuli below the injury level (commonly referred to as autonomic dysreflexia; 24), may also provoke periods of GI hypoxia due to hyperreactivity of the mesenteric bed (47). Although the mechanism remains incompletely understood, the impaired GI blood flow we have observed and mesenteric hyperreactivity as is likely to occur during autonomic dysreflexia may contribute to the chronic gastrointestinal dysfunction experienced by individuals with SCI (3, 12, 14, 37, 53, 56, 70).

Perspectives and Significance

Our novel data reveal that basal mesenteric blood flow is markedly diminished following a severe spinal cord injury at spinal T3. Furthermore, postprandial splanchnic vascular reflexes are blunted following experimental T3-SCI. We propose that changes in nutrient-vascular relationships may render the post-SCI gut susceptible to episodic ischemic and inflammatory events. On the basis of clinical reports, we further propose that these changes in nutrient-vascular relationships may last for weeks after the original SCI and that these comorbidities may contribute to the GI dysfunction observed in the SCI population.

GRANTS

This work was supported by National Institute of Neurological Disorders and Stroke (NINDS) Grants R01-NS-049177 (G. M. Holmes) and F31-NS-087834 (E. M. Swartz). Training Fellowship support for N. Pironi was provided by American Heart Association (12UEFL1008000, S. D. Stocker).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

E.M.B. and G.M.H. designed the study; E.M.B., N.P., and G.M.H. performed in vivo studies; G.M.D. performed qRT-PCR and analysis; T.K.C. scored histological specimens. E.M.B. and G.M.H. prepared figures; E.M.B. and G.M.H. drafted and revised the manuscript with input from all authors; E.M.B. and G.M.H. approved final version of manuscript.