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(Stroke. 1996;27:1850-1858.)
© 1996 American Heart Association, Inc.

Articles

Effect of Proximal Arterial Perfusion Pressure on Function, Spinal Cord Blood Flow, and Histopathologic Changes After Increasing Intervals of Aortic Occlusion in the Rat

Yutaka Taira, MD, PhD Martin Marsala, MD

the Department of Anesthesiology, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan (Y.T.), and Anesthesiology Research Laboratory, University of California, San Diego (M.M.).

Correspondence to Martin Marsala, MD, PhD, Anesthesiology Research Laboratory, 0818, University of California, 9500 Glimmer Dr, San Diego, CA 92093. E-mail mmarsala@ucsd.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Background and Purpose Cross-clamping of the thoracic aorta results in spinal cord ischemia and prominent systemic hypertension. Using a rat model of transient spinal cord ischemia, we examined the effects of manipulation of proximal aortic blood pressure on spinal cord blood flow (SCBF), neurological dysfunction, and changes in spinal histopathology after increasing intervals of aortic occlusion.

Methods Aortic occlusion was induced by the inflation of a 2F Fogarty catheter placed into the thoracic aorta in rats anesthetized with halothane (1.5%). A tail artery was cannulated to monitor distal arterial pressure (DAP). To measure SCBF, a laser probe was implanted into the epidural space of the L-2 vertebra. To manipulate proximal arterial pressure (PAP), the left carotid artery was cannulated with a 20-gauge polytetrafluoroethylene catheter to permit blood withdrawal and infusion from a peripheral reservoir during aortic occlusion. In a survey study, spinal cord ischemia was induced in single animals at intervals of 6, 10, 15, 30, or 40 minutes with PAP controlled at 40, 60, 80, and 110 to 120 mm Hg. In a second series, ischemia was induced in groups of animals for 0, 6, 8, 10, and 12 minutes with PAP controlled at 40 mm Hg. After ischemia the animals survived for 2 to 3 days. During this recovery period, neurological functions were evaluated, followed by quantitative histopathology of the spinal cord.

Results Under normal conditions, cross-clamping yields an acute proximal hypertension (125 to 135 mm Hg), a fall of DAP to 15 to 22 mm Hg, and a decrease in SCBF to 7% to 11% of baseline values. With the use of the external reservoir, proximal hypertension could be abolished and the PAP maintained at target pressures. In these studies a typical syndrome of tactile allodynia, spastic paraplegia, and necrotic changes affecting the central part of the gray matter after 24 to 48 hours of reperfusion was observed at the following combinations of ischemic intervals and PAP values: >10 minutes/40 mm Hg; >12 minutes/60 mm Hg; >16 minutes/80 mm Hg; and >30 minutes/uncontrolled. Lowering PAP resulted in a corresponding decrease in residual SCBF. Systematic studies at a PAP of 40 mm Hg at occlusion intervals of 6, 8, 10, and 12 minutes revealed that 100% of rats were paraplegic after 10- and 12-minute ischemia, and these rats showed corresponding signs of spinal histopathology.

Conclusions The present study shows that systemic intraischemic hypotension (40 mm Hg) significantly potentiates neurological dysfunction after transient aortic occlusion. The mechanism of the observed effect may include elimination of collateral flow during aortic occlusion and/or consequent potentiation of hypoperfusion during reperfusion. These data indicate that PAP during occlusion should be monitored and/or controlled because it is a critical variable in the determination of outcome in this model of spinal cord ischemia.

Key Words: hypotension • ischemia • paraplegia • spinal cord • rats

	Introduction

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Cross-clamping of the thoracic aorta for extended intervals leads to irreversible spinal cord neuronal damage and loss of neurological function.¹ The extended interval (typically >30 to 60 minutes depending on the experimental species and model) necessary for such irreversible changes to be observed emphasizes that such cross-clamping results in a low (nonzero) flow state. This is consistent with the anatomy of the spinal vasculature. The spinal cord receives a major portion of nutritive perfusion from the segmental radicular arteries arising from the descending aorta and from the vertebral arteries, which provide a significant flow into the anterior spinal artery at the cervical and upper thoracic levels.² The magnitude of the insult to spinal function will thus be altered by factors that alter the perfusion through the collateral circulation. One important determinant that will influence collateral flow is proximal arterial perfusion pressure (PAP). Aortic cross-clamping will induce significant hypertension proximal to the cross-clamp. To protect the nonclamped cranial circulation and reduce cardiac overload, efforts may be made to reduce cardiac output or reduce peripheral vascular resistance.^{3 4}

A variety of animal models have been described to study the effects of cross-clamping, including dog,⁵ rabbit,⁶ baboon,⁷ pig,⁸ and rat.⁹ In these models the thoracic or abdominal aorta is occluded, causing significant but incomplete reduction in spinal cord blood flow (SCBF). However, the effects of PAP have not been systematically investigated in these models.

We have characterized a minimally invasive rodent model in which aortic occlusion is achieved by the placement and subsequent inflation of a 2F Fogarty catheter in the descending thoracic aorta.^{10 11 12} In previous work with this model, we have shown that aortic occlusion results in a significant reduction in lumbar SCBF to 8% to 12% of control as defined by laser-Doppler flowmetry and that 20 to 30 minutes of occlusion represents a critical interval associated with the development of spastic or flaccid paraplegia. This relatively prolonged occlusion time required to evoke neurological dysfunction indicates the presence of an effective collateral system that will preserve sufficient SCBF for relatively prolonged periods after aortic occlusion. Importantly, we have noted, as have others,¹³ that the occlusion is typically accompanied by an increase in proximal blood pressure (ie, above the level of the aortic occlusion).

By using the aforementioned rat model in the present study, we sought to characterize the effect of controlled (40 to 120 mm Hg) PAP on time-dependent changes in SCBF (as measured by laser-Doppler flowmetry), the development of neurological dysfunction, and corresponding changes in spinal histopathology.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
All work described in this study was performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of California, San Diego.

General Preparation
Male Sprague-Dawley rats (n=56; weight, 350 to 375 g) were used in this study. Animals were anesthetized in an acrylic plastic box with 4% halothane in room air. After induction, rats were maintained with 1% to 1.5% halothane in an air/O₂ mixture delivered by an inhalation mask. Body temperature was monitored with a rectal probe inserted 8 cm into the rectum and maintained between 37.2°C and 37.5°C with an underbody heating pad.¹⁴ The tail artery was cannulated with a 22-gauge polytetrafluoroethylene catheter for monitoring distal arterial pressure (DAP) and for intra-arterial infusion of heparin. The left carotid artery was cannulated with a 20-gauge polytetrafluoroethylene catheter for monitoring PAP and withdrawing blood (Fig 1). For induction of spinal cord ischemia, the left femoral artery was exposed, and a 2F Fogarty catheter (model CV 1035, American V. Mueller) was passed to the thoracic aorta so that the tip of the catheter reached the left subclavian artery.¹¹ On the basis of preliminary dissections, this level corresponded to a distance of 10.8 to 11.4 cm from the site of insertion. In addition to occlusion of the descending aorta, inflation of the balloon with 0.05 mL of saline has been found to also directly occlude one to two upper thoracic radicular arteries branching from the aorta at this level. Immediately after completion of arterial cannulations, all animals received 200 U of heparin (0.2 mL) injected into the tail artery.

View larger version (37K): [in this window] [in a new window]   Figure 1. Experimental system. Under continuous monitoring of proximal and distal arterial blood pressure, blood is withdrawn into the blood collection circuit (BCC; 37°C) during balloon-induced aortic occlusion. Note the presence of collateral arteries (internal thoracic artery [ITA] and vertebral artery [VA]), which may modulate spinal cord blood flow during occlusion. ASA indicates anterior spinal artery; FA, femoral artery; LCA, left carotid artery; RA, radicular artery; SCA, subclavian artery; and TA, tail artery.

Induction of Spinal Cord Ischemia and Control of PAP
To induce spinal cord ischemia, the balloon was inflated with 0.05 mL of saline. The efficiency of the occlusion was evidenced by an immediate and sustained loss of any detectable pulse pressure and by a drop in the DAP measured in the tail artery. To control the PAP during occlusion, the blood from the carotid artery was allowed to flow into a collecting circuit filled with heparinized saline (4 U/mL of saline) and positioned at various heights measured from the level of the animal's body. These heights corresponded to the targeted PAP (ie, 40 mm Hg=54 cm; 60 mm Hg=81 cm; 80 mm Hg=108 cm; 90 mm Hg=uncontrolled). The temperature of the blood collection circuit was maintained at 37°C. After ischemia, the balloon was deflated, and the collected blood was reinfused by the addition of 100 mm Hg of pressure to the blood-collecting syringe during a 60-second period. Stabilization of the arterial blood pressure was then monitored for an additional 10 minutes, after which arterial lines were removed, and the wounds were closed. After completion of all surgical procedures (10 minutes after reperfusion), 0.4 mL of protamine sulfate (4 mg) was administered intraperitoneally. Animals were then returned to their cages and assessed for recovery of neurological function for 24 to 72 hours. For ethical reasons, animals suffering from acute paraplegia with no sign of recovery after 48 hours were killed and perfusion-fixed for histopathologic analysis. Animals that had no neurological deficit were allowed to survive for 3 days and then perfusion fixed.

Measurement of SCBF (n=4)
To assess the relationship between PAP, DAP, and SCBF during occlusion, a separate group of animals (n=4) was used. To measure SCBF, the L-2 vertebra was exposed by midline incision and blunt dissection. A small hole was then drilled on the right side of the lamina overlying the L-4 and L-5 spinal segments. The tip of a laser probe (0.8 mm in diameter; model BPM 403, Laserflo blood perfusion monitor, TSI Inc) was placed into the epidural space by a supportive polytetrafluoroethylene tube attached to the bone with dental cement. SCBF was then continuously monitored before and during ischemia with a 5-second averaging cycle. After the baseline data were obtained, spinal cord ischemia was induced as described above. During aortic occlusion, the PAP was gradually decreased at 3-minute intervals, as follows: 0 to 3 minutes, uncontrolled; 3 to 6 minutes, 100 mm Hg; 6 to 9 minutes, 80 mm Hg; 9 to 12 minutes, 60 mm Hg; and 12 to 15 minutes, 40 mm Hg). During the entire occlusion period (0 to 15 minutes), the laser-Doppler signal and the corresponding PAP and DAP were recorded. At the end of the occlusion period, the rats were killed by deep halothane anesthesia, and "zero" flow data were obtained.

Ischemia Paradigms (n=52)
The study was divided into two independent parts: a survey and a systematic assessment.

Ischemic Interval/PAP Survey
To obtain behavioral data on the effect of controlled PAP versus increasing intervals of ischemia, the animals were exposed to intervals ranging from 6 to 40 minutes of ischemia with the PAP controlled at 40, 60, 80, and 120 mm Hg. A single animal was used for one ischemic interval and PAP. After ischemia, the animals were allowed to recover for 2 to 3 days. Recovery of neurological function was periodically assessed, and after 2 to 3 days of survival all animals were killed.

Systematic Assessment
On the basis of the experimental data obtained in the preceding survey study, an additional group of animals (n=24) was used. Rats were divided into four individual groups (n=6 in each) and subjected to 6, 8, 10, and 12 minutes of ischemia with PAP controlled at 40 mm Hg. We chose this level of systemic hypotension (ie, 40 mm Hg) based on our initial SCBF and behavioral survey study, which showed that such a degree of systemic hypotension is sufficiently effective to eliminate a gradual increase in DAP after aortic occlusion and provides a reliable neurological deficit after a relatively short (10-minute) occlusion interval. After ischemia, animals survived for 2 to 3 days, and the neurological outcome was assessed. In control animals, the balloon catheter was placed in the thoracic aorta but not inflated, and systemic arterial pressure was decreased to 40 mm Hg for 12 minutes (ie, the maximum interval at this pressure). At the end of the survival period, all animals were perfusion fixed for histopathologic examination of the spinal cord (see below).

Behavioral Assessment
During reperfusion (1, 2, 4, 8, 24, 48, and 72 hours), recovery of motor and sensory functions was assessed with the use of the following grading systems.

Motor Function
Motor function was quantified by assessment of ambulation and placing and stepping responses. Ambulation (walking with lower extremities) was graded as follows: 0, normal (symmetrical and coordinated ambulation); 1, toes flat under body when walking but ataxia present; 2, knuckle-walking; 3, movement in lower extremities but unable to knuckle-walk; and 4, no movement, drags lower extremities. We assessed the placing/stepping reflex by dragging the dorsum of the hind paw over the edge of a surface. This normally evokes a coordinating lifting and placing response (eg, stepping), which was graded as follows: 0, normal; 1, weak; and 2, no stepping. A motor deficit index was calculated for each rat at each time interval. The final index was the sum of the scores (walking with lower extremities and placing/stepping reflex). The maximum deficit was indicated by a score of 6.

Sensory Function
Reaction to hind paw pinch was graded as follows: 0, normal response; 1, some response present; and 2, no response to pinch. Allodynia was defined as vigorous squeaking and agitation in response to light stroking of the flank.

Histopathology
At the end of the survival period, rats were terminally anesthetized with pentobarbital (100 mg/kg IP) and phenytoin (25 mg/kg IP). Each rat was then transcardially perfused with 100 mL of heparinized saline followed by 150 mL of 4% paraformaldehyde in phosphate buffer (pH 7.4). Twenty-four hours later, the spinal cords were removed and postfixed in the same fixative for 2 to 14 days. After this period, the spinal cords were removed and the L-3 to L-5 spinal segments dissected. Samples were then postfixed in 1% buffered OsO₄ and embedded in high-viscosity epoxy resin (Araldite, Ciba-Geigy Corp). Semithin sections (1 µm thick) were prepared and stained with p-phenylenediamine.

Five representative sections taken from L-3 to L-5 segments were coded in each animal and then subjected to a systematic examination. The degree of gray matter damage (necrosis or dark neurons) was then judged individually for each of three regions: region I, damage involving Rexed laminae I to VI; region II, damage involving laminae VII and X; and region III, damage involving laminae VIII and IX. Each region was scored according to the following criteria: 0, normal, no detectable changes; 1, damage affecting <10% of scored area; 2, damage affecting 10% to 50% of scored area; and 3, damage affecting >50% of scored area.

Average values from five sections and three laminar levels were then calculated so that the final score for one spinal cord ranged between 0 (no detectable changes) and 9 (>50% damage). Scores were tabulated and the analysis was prepared by the observer (M.M.) without knowledge of the behavioral outcome or the duration of occlusion.

Statistical Analysis
Statistical analysis of physiological data was performed by one-way ANOVA for multiple comparisons followed by appropriate post hoc tests (Fisher's, Scheffe's F, and Dunnett's tests). For analysis of neurological outcome in the individual occlusion group, significant overall values were obtained with a Kruskal-Wallis test. Specific comparisons between experimental groups were performed with the Mann-Whitney U test for independent means. Examination of the covariance between PAP, DAP, and SCBF was performed with Spearman's rank correlation test. A value of P<.05 was considered significant. Data were expressed as mean±SD.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Physiological Variables
Baseline body temperature ranged between 36.7°C and 37.3°C, and DAP was 95 to 103 mm Hg. No significant differences between experimental groups were detected (Table 1).

View this table: [in this window] [in a new window]   Table 1. Physiological Data in Animals Subjected to Increasing Intervals of Ischemia With Mean Proximal Arterial Pressure Controlled at 40 mm Hg

Survey of Effect of Controlled PAP on DAP, SCBF, and Neurological Outcome
Blood Pressure and SCBF
A clear correlation between PAP and the magnitude of the reduction of DAP and SCBF after aortic occlusion was observed. In animals with uncontrolled PAP (PAP of 127±8 mm Hg during occlusion), DAP was 19±4 mm Hg during 10 to 40 minutes of aortic occlusion (Fig 2A, Tables 1 and 2) but decreased to 7±1 mm Hg when PAP was controlled at 40 mm Hg (Fig 2B, Tables 1 and 2). Stepwise decreases in PAP after aortic occlusion caused a gradual decrease in SCBF ranging from 8±4% of baseline in animals with uncontrolled PAP to 2.5±1.6% in the 40 mm Hg group and dropped to 0.9±0.6% after induction of cardiac arrest (Table 2, Fig 3).

View larger version (28K): [in this window] [in a new window]   Figure 2. Proximal (PAP) and distal (DAP) arterial pressure in rats subjected to aortic occlusion with uncontrolled PAP (A) or PAP controlled at 40 mm Hg (B). Note apparent higher decrease in DAP during occlusion in animals with DAP controlled at 40 mm Hg.

View this table: [in this window] [in a new window]   Table 2. Effect of Controlled Proximal Arterial Pressure on Distal Arterial Pressure and Spinal Cord Blood Flow During Occlusion and Corresponding Duration of Critical Ischemic Time

View larger version (39K): [in this window] [in a new window]   Figure 3. Effect of mean proximal arterial pressure (MPAP) manipulation on mean distal arterial pressure (MDAP) and spinal cord blood flow (SCBF) during aortic occlusion. Note a significant reduction in MDAP (A) and SCBF (B) with reduction of PAP. A, Correlation between MPAP and MDAP; B, correlation between SCBF and MPAP; and C, correlation between SCBF and MDAP. A highly significant correlation between the magnitude of PAP reduction and SCBF was observed. CA indicates cardiac arrest; CONT, controlled.

Neurological Outcome
Table 3 presents neurological outcome in individual animals from the survey portion of the study. As indicated, there was an increasing incidence of motor dysfunction with an increasing interval of ischemia and decreasing PAP during periods of occlusion. Thus, the acute type of paraplegia in animals with uncontrolled PAP was observed only after 40 minutes of aortic occlusion, while a comparable neurological deficit developed after 10 minutes of aortic occlusion when PAP was controlled at 40 mm Hg. A delayed paraplegia was observed in some rats. These animals typically displayed full recovery of function between 12 and 24 hours after ischemia but then gradually developed a spastic type of paraplegia over 1 to 3 days.

View this table: [in this window] [in a new window]   Table 3. Distributional Pattern of Neurological Outcome: Survey of Duration of Ischemia vs Proximal Blood Pressure

Neurological Outcome With Increasing Intervals of Occlusion at PAP of 40 mm Hg: Systematic Assessment (Figs 4 and 5)
On the basis of these survey behavioral data, rats were entered into groups that underwent aortic occlusions for intervals of 0, 6, 8, 10, and 12 minutes (n=6 in each group) at a PAP of 40 mm Hg.

View larger version (35K): [in this window] [in a new window]   Figure 4. Motor deficit index assessed at 1 to 72 hours in animals after 6, 8, 10, and 12 minutes of aortic occlusion with proximal arterial pressure controlled at 40 mm Hg during aortic occlusion. No detectable effect in the 6-minute occlusion group was seen, while a high incidence of acute paraplegia after 10 and 12 minutes of aortic occlusion was observed. Symbols as in Fig 3.

View larger version (45K): [in this window] [in a new window]   Figure 5. Sensory function assessed at 1 to 72 hours in animals after 6, 8, 10, and 12 minutes of aortic occlusion with proximal arterial pressure controlled at 40 mm Hg during aortic occlusion. Note the increased incidence of complete sensory loss with increased intervals of ischemia.

In the control (nonocclusion) group, no detectable neurological dysfunction was seen at a PAP of 40 mm Hg for 12 minutes during the 72 hours of survival, and all animals displayed preserved motor and sensory functions (data not shown).

After 6 minutes of ischemia, slight muscle weakness affecting lower extremities (motor deficit index=1) was seen in several animals during the 72 hours after ischemia; however, all animals were able to walk and step, and the total neurological score was not significantly different compared with control nonischemic animals. Two animals showed transient hypesthesia.

In the group with 8 minutes of ischemia, the majority of animals showed a moderate motor deficit (ataxia or knuckle-walking) for the initial 8 hours of reperfusion. During the subsequent 24 hours, 2 animals developed spastic paraplegia and 4 others recovered and displayed normal ambulation. The 2 paraplegic animals also showed a complete loss of sensory function.

In the group with 10 minutes of ischemia, 5 of 6 animals had an acute spastic type of paraplegia with no signs of recovery for the additional 2 days of survival. One animal had delayed paraplegia at 24 hours. All animals displayed sensory dysfunction. Four animals showed partial loss of sensory response, and 2 animals displayed allodynia.

In the group with 12 minutes of ischemia, all animals (6 of 6) displayed acute and persistent spastic paraplegia, which remained unchanged for 2 days. At 24 hours, 4 animals had complete sensory loss, 1 had hypesthesia, and 1 displayed allodynia.

Histopathologic Changes
A clear correlation between the degree of histopathologic damage and the duration of aortic occlusion was observed (Fig 6).

View larger version (31K): [in this window] [in a new window]   Figure 6. Individual neuropathology score (0, no damage; 9, >50% of gray matter damage) after 6, 8, 10, and 12 minutes of aortic occlusion with proximal arterial pressure controlled at 40 mm Hg. Note the presence of extensive neuropathological damage and corresponding loss of motor function in animals subjected to 10 and 12 minutes of aortic occlusion. *P<.05, significantly different compared with 6-minute group. The symbol indicates walking animals; , paraplegic animals.

In the control nonischemic group, animals displayed full recovery of motor and sensory functions with no histopathologic changes in the spinal cord sections taken from L-2 to L-5 segments. All neuronal elements including small and medium-sized interneurons and large -motoneurons survived without noticeable changes. Comparably, no detectable changes in neuropils were observed (Fig 7A).

View larger version (509K): [in this window] [in a new window]   Figure 7. A, Transverse semithin (1 µm) section taken from L-3 spinal segment of a control nonischemic animal that survived for 3 days after systemic blood pressure was controlled at 40 mm Hg for 12 minutes. Note the presence of normally appearing medium-sized and large interneurons (lamina VII) as well as neuropil (p-phenylenediamine stain; original magnification x10). LVII indicates lamina VII. B, Transverse semithin (1 µm) section taken from the L-3 spinal segment of an animal that survived for 3 days with no signs of neurological deficit after 8 minutes of aortic occlusion. Note dark-staining -motoneurons (arrowheads) localized close to normal-appearing neurons (p-phenylenediamine stain; original magnification x20). C, Transverse semithin (1 µm) section taken from the L-3 spinal segment of an animal that survived for 2 days with fully developed spastic paraplegia after 10 minutes of aortic occlusion. Note the presence of extensive necrotic changes and neuropil cavitation localized in lamina VII. Relatively well-preserved gray matter in the peripheral part of the ventral horn (lamina VIII) can also be seen (p-phenylenediamine stain; original magnification x10). D, L-3 spinal segment of an animal that survived for 2 days with fully developed spastic paraplegia after 10 minutes of aortic occlusion. A group of normal-appearing -motoneurons (arrowheads) can be seen (p-phenylenediamine stain; original magnification x40).

In the groups with 6 or 8 minutes of ischemia, in animals that showed full recovery of neurological function, occasional neuronal degeneration expressed as the presence of "dark" and/or shrunken neurons was observed. These neurons were typically localized in the central part of the intermediate zone (lamina VII) and the dorsomedial part of the dorsal horn (Fig 7B). However, the majority of interneurons and -motoneurons showed no detectable changes. In two animals that displayed a delayed paraplegia (8-minute occlusion group), histopathologic changes that resembled those seen in groups with 10 or 12 minutes of ischemia were observed (see below).

In the groups with 10 or 12 minutes of ischemia, histopathologic changes in the animals with a fully developed spastic type of paraplegia were characterized by the presence of extensive necrotic changes and irregular cavitation of the gray matter distributed between laminae III to VII in the L-2 to L-5 segments. Despite these extensive and advanced histopathologic changes, neuronal pools localized in the peripheral part of the gray matter, ie, laminae I to II, lamina X, and laminae VIII to IX, showed near-normal structure (Fig 7C). Similarly, corresponding with the presence of spasticity, the majority of -motoneurons showed normal structure with a centrally localized nucleus and nucleolus (Fig 7D).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
Transient spinal cord ischemia and the resulting loss of neurological function represent a serious consequence of thoracoabdominal aortic resection.¹ The main pathophysiological factor determining recovery of neurological function after ischemia appears to be the magnitude of the reduction of SCBF during aortic occlusion. Once the lack of oxygen and glucose reaches a critical level, it subsequently triggers a chain of events initially characterized by depletion of high-energy substrate in the spinal gray matter¹⁵ and loss of evoked spinal cord neuronal activity,¹⁶ followed by anoxic depolarization of neurons and significant increase in the extracellular concentration of several active factors, including excitatory amino acids,^{12 17} prostaglandins,¹⁸ and/or oxygen free radicals.¹⁹ The onset of the ischemic state depends on the termination of sufficient nutritive perfusion. In the case of aortic aneurysm repair, it appears certain that the cord behaves as if it is in a low-flow state. Since the human spinal cord has a heterosegmental blood supply² (see below), the residual flow must arise from so-called collateral vascularization. Maintenance of that collateral perfusion is likely an important variable in determining cord survival during an extensive interval of thoracic aortic cross-clamping. In the present study we considered the role of arterial perfusion pressure on behavior and histopathologic outcome in a well-defined, minimally invasive, rat model of transient aortic occlusion.

Rat Spinal Cord Vasculature and Collateral System
The rat, as in several larger animal species such as monkey, dog, and cat, possesses a heterosegmental aorta with 8 to 14 radicular arteries that give rise to one anterior and two posterior spinal arteries. Interestingly, there is a significant difference between the size and the localization of the radicular arteries, with the major artery (Adamkiewicz artery) typically branching from the thoracoabdominal aorta at the T-8 to L-2 segmental levels.²⁰ Although the occlusion of the thoracic aorta substantially reduces the nutritive flow below the level of the occlusion, several collateral systems may significantly contribute to the preservation of the distal flow, particularly after single aortic cross-clamp. One of the major contributors may derive from the internal thoracic artery, which branches from the subclavian arteries and has well-developed anastomoses with the lower thoracic arterial system and may provide a retrograde flow to the segmental spinal cord vessels. Second, a continuing collateral flow down through the ventral chest wall and the abdominal free wall anastomosing with the iliac arteries could also contribute to a gradual increase in DAP after aortic occlusion. Third, both subclavian arteries supplying the vertebral artery may provide additional flow through the anterior spinal artery.⁹ These properties are remarkably similar to the spinal vascular organization present in humans.² This similarity suggests that the rat model is an appropriate preparation for characterizing factors influencing spinal cord survival in aortic cross-clamping.

Effect of Controlled PAP on DAP and SCBF
One of the experimental approaches to eliminating or minimizing the effect of collateral flow is to reduce PAP during the ischemic period. This can be achieved (1) pharmacologically by systemic delivery of vasodilatatory agents such as nitroprusside and/or (2) by removal of a portion of the circulation blood (ie, induction of hypovolemic hypotension). Both approaches have been successfully used to control the degree of ischemia in models of brain and spinal cord ischemia.^{4 13 21}

In the present study systemic pressure was controlled during cross-clamping by withdrawal of arterial blood from the left carotid artery during aortic occlusion. Concurrent measurements of DAP and SCBF showed a significant correlation between the magnitude of the reduction of DAP and SCBF, which was proportional to the PAP during occlusion. Importantly, in animals with uncontrolled PAP, a progressive increase in DAP was observed when DAP initially dropped after occlusion to approximately 9 to 14 mm Hg and then gradually increased during occlusion to 20 to 22 mm Hg.

One important question relates to the mechanism of the observed gradual increase in DAP. In the present study a balloon catheter inflated with 0.05 mL of saline was used to occlude the descending thoracic aorta. We have observed that such a volume of balloon inflation provides complete occlusion of the aortic lumen, thus eliminating the possibility that there is persisting residual flow around the balloon. Based on this reasoning, we believe that under the condition of fixed aortic occlusion the gradual increase in DAP likely reflects a gradual opening of collaterals and corresponding increases in distal arterial blood flow.

These data also indicate that the collateral flow is functionally not static but may, as a result of increased PAP, increase the amount of blood flow below the occlusion and thus significantly contribute to spinal cord perfusion during aortic occlusion.

However, when the PAP was controlled at 40 mm Hg, an immediate and persistent decrease in DAP was observed after balloon inflation. Here, DAP ranged between 6 and 7 mm Hg during the period of occlusion with no observable fluctuations. The magnitude of reduction in DAP in these animals was almost identical to the levels observed after cardiac arrest (4.7 mm Hg), suggesting near total elimination of collateral flow.

As for DAP, with a gradual decrease of PAP, SCBF displayed a progressive reduction during the occlusion, with the lowest values (2.5% of baseline flow) observed in the 40 mm Hg group. As defined in the behavioral part of the study, such a reduction in SCBF, if it lasted 10 to 12 minutes, was associated with a high incidence of acute paraplegia.

As described above, the manipulation of systemic arterial pressure and corresponding failure of collaterals to open during aortic occlusion appears to be a primary variable that will determine outcome in this experimental model. However, we note that subsequent hemodynamic conditions that develop during reperfusion may also be relevant. It has been shown that after transient spinal ischemia, spinal cord hyperemia is followed by hypoperfusion. It has also been demonstrated that the magnitude of postischemic hyperemia, consequent hypoperfusion, and corresponding neurological outcome depend on the duration and/or completeness of spinal ischemia.^{22 23 24} Although not determined in the present study, it is likely that the magnitude of spinal cord hypoperfusion after ischemia could be substantially potentiated after the completeness of spinal ischemia is increased (ie, as with decreasing PAP), and this could directly influence neurological outcome.

Neurological Outcome
In the present study a clear correlation between duration of ischemia, magnitude of reduction of PAP during occlusion, and recovery of neurological function was observed. Thus, animals with PAP controlled at 40 mm Hg consistently displayed paraplegia after 10 minutes of aortic occlusion, whereas animals with uncontrolled PAP (ie, PAP of 127 mm Hg during occlusion) showed comparable deficit only after 30 to 40 minutes of aortic occlusion. No acute paraplegia was observed after 8 minutes of occlusion; instead, two rats displayed a delayed manifestation of neurological dysfunction. A comparable, progressively developing deficit in a rabbit spinal cord ischemic model has been described.⁶ The above data suggest that in the present model, the ischemic threshold (ie, the period causing acute paraplegia) can be relatively precisely defined.

Histopathologic Changes
Two important points should be noted. First, at a PAP of 40 mm Hg, occlusion intervals of 8 minutes or less resulted in minimal changes in histopathology. In contrast, at intervals of 10 minutes or greater, there was a precipitous increase in the incidence of dark-staining neurons and necrotic changes in the lumbar spinal cord segments. This tight grouping emphasizes the reduction in variability that can accrue from control of PAP. Second, we noted a strong correlation between changes in histopathology and the observed neurological deficit. Thus, at ischemic intervals of 6 and 8 minutes with a PAP of 40 mm Hg, we found minimal neurological signs in terms of either sensory or motor indices. In contrast, at 10 and 12 minutes of occlusion, animals with acute-onset paraplegia showed the highest degree of histopathologic changes in the L-2 to L-5 spinal segments, as analyzed 2 days after reperfusion. These effects were manifested as the presence of dark shrunken neurons and/or necrotic foci typically localized between laminae III and VII. Consistent with the presence of spastic paraplegia, the majority of these animals showed a predominance of normally appearing -motoneurons in laminae VIII and IX. These histopathologic data are similar to those seen in paraplegic dog,^{25 26 27} cat,²⁸ and rabbit.^{6 14 17}

Significance
Control of PAP appears to be as important as control of temperature in terms of the effects of various treatments on outcome in aortic cross-clamp studies. Drug treatments that result in hypertension may diminish the impact of cross-clamping, whereas those that lower PAP may have no effect on outcome or may worsen outcome despite a protective mechanism if the treatment results in simple hypotension. At a minimum, both temperature and PAP must be controlled in mechanistic studies.

Conclusions
Data from the present study show that increased PAP significantly potentiates the effectiveness of collateral flow and improves SCBF during aortic occlusion. This effect can be nearly eliminated by decreasing systemic blood pressure to 40 mm Hg during occlusion. Consistent with the completeness of spinal cord ischemia, these animals also showed a high incidence of spastic paraplegia even after relatively short (10-minute) intervals of aortic occlusion. These data also indicate that PAP during occlusion should be monitored and/or controlled because it is a critical hemodynamic variable in the determination of motor and sensory outcomes in this model of spinal cord ischemia.

	Acknowledgments

 
This study was supported by grant NS32794 from the National Institutes of Health (Dr Marsala).We express our thanks to Tony L. Yaksh, PhD, and David Dirig, Anesthesiology Research Laboratory, University of California, San Diego, for their helpful advice in the preparation of this manuscript. We are also grateful to Shelle A. Malkmus, AHT, for excellent technical assistance.

Received January 2, 1996; revision received April 22, 1996; accepted May 3, 1996.

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Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 

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Editorial Comment

William I. Rosenblum, MD, Guest Editor

Division of NeuropathologyMedical College of VirginiaVirginia Commonwealth UniversityRichmond, Va

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction

 
The accompanying article concerns an important and often neglected subject: ischemic injury of the spinal cord. The authors have tried to develop an experimental model that will produce a consistent morphological representation. The authors explain that to achieve this goal, the perfusion pressure through collaterals must be maintained. Otherwise, presumably variable degrees of suboptimal pressure and flow through the collaterals lead to a variable degree of infarction. This study is of interest because it provides the experimentalist with a useful model and because it illustrates, albeit indirectly, the importance of collateral flow in the event of occlusion of a major vessel supplying the spinal cord.

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