Intravenous Infusion of Magnesium Chloride Improves Epicenter Blood Flow during the Acute Stage of Contusive Spinal Cord Injury in Rats

Animals and experimental design

Seventy-eight young adult female Sprague Dawley rats were used (180–220 g; Harlan; Table 1). We first determined the time course of vascular changes between 20 min and 48 h following a contusive SCI. A second experiment determined the effects of intravenous infusions of magnesium over a 24 or 48 h period. Third, we tested whether a 7 day magnesium infusion would be neuroprotective and improve locomotor function over 4 weeks. All animal procedures were conducted according to the guidelines of National Institutes of Health, and were approved by the University of Louisville Institutional Animal Care and Use Committee. Animals were allowed to habituate to their cages for at least 48 h after arrival, and had free access to food and water. Investigators involved in surgeries, behavioral testing, and quantification of histological results were blinded to the treatment group.

SCI

All invasive procedures were conducted with the animals under deep anesthesia with an intramuscular injection of a 3.3 mL/kg mixture containing 25 mg/mL ketamine hydrochloride (Abbott Laboratories, Abbott Park, IL), 1.2 mg/mL acepromazine maleate (The Butler Company, Dublin, OH), and 0.25 mg/mL xylazine (The Butler Company) in 0.9% saline. Animals received a subcutaneous injection of 5 mL of saline for rehydration, eye protective ointment (Rugby Laboratories, Duluth, GA), and an intramuscular injection of 0.1 mL of a 100 μg/mL stock solution of gentamicin (ButlerSchein, Dublin, OH) to reduce infections.

All surgeries were performed under aseptic conditions and under visualization with a Wild M695 motorized surgical microscope. After a dorsal skin incision and soft tissue dissection at the T8-T10 level, the T9 vertebra was stabilized in a frame with steel clamps inserted under the transverse processes. Laminectomy was performed without opening the dura. The spinal cord was contused using an Infinite Horizons (IH) impactor (Precision Systems and Instrumentation, Lexington, KY) set at 150 kdyn with a 2.5 mm diameter impactor tip. Afterwards, the muscles were sutured with 5.0 silk and the skin was held with 9 mm metal clips (Braintree, ACS-cs, Braintree, MA). Bacitracin ointment (Qualitest Pharmaceuticals, Huntsville, AL) was applied to the wound to reduce infections, and another 5 mL of normal saline solution was injected subcutaneously. The animals were allowed to recover on a water-circulating heating pad at 37°C (Gaymar Industries, Orchard Park, NY). Sham-operated animals received only a laminectomy. Postoperative care included twice daily bladder expression.

Spinal cord laser Doppler measurements

To measure spinal cord blood flow we used Doppler laser measurements in four rats with a Moor VMS-LDF1 device (Moor instruments Inc., Wilmington, DE) with a VP10M200ST master probe connected to a P10D plastic fiber (outer diameter 0.5 mm). After vertebral stabilization and a T9 laminectomy, the probe was placed ∼1 mm above the cord, close to the rostral end of laminectomy window, which was covered with a layer of lucent water-based gel (Surgilube, Fougera & Co, Melville, NY). The laser beam covered an area of 1 mm² and penetrated up to 1 mm into the tissue, thus covering more than half of the depth of cord and obtaining readings from the superficial vessels as well as much of the gray matter where microvascular perfusion is most abundant. Ten baseline readings were obtained before the contusion injury. Afterwards, the probe was removed and a 150 kdyn force injury was performed as described previously. Spinal cord blood flow was repeatedly measured in each animal at various times between 1 min and 48 h after the injury, positioning the laser Doppler probe to the initial measurement area. The bony edge of the laminectomy window was used as a reference point to ensure the same position of the optic probe in repetitive measurements. For measurements at intervals> 1 h, animals were anesthetized with Isoflurane (Butler, Dublin, OH). An average of 10 readings at each given time point was calculated for each animal, and expressed as a percentage of baseline readings.

Continuous intravenous infusions

Animals received a continuous intravenous infusion of phosphate-buffered saline (PBS) or MgCl₂ (31 mg/day=155 mg/kg/day in ddH₂O; Cat #M2670; Sigma, St. Louis, MO). Earlier studies had used the magnesium sulfate form by itself. ^17,18 However, because of slightly better outcomes after MgCl₂ than after magnesium sulfate in combinations with polyethylene glycol following a contusion injury, MgCl₂ had been chosen for further optimization and characterization (combined with polyethylene glycol). ^18,30 Therefore, we chose to test MgCl₂. Both the chloride and sulfate forms are used clinically. The daily dose of 760 μmol/kg was extrapolated from a previous study in which 190 μmol/kg per intravenous injection was deemed effective and in line with tolerable doses in humans, and a 6 h interval (four injections per 24 h) protected most tissue. ¹⁸ This dose also showed efficacy when combined with polyethylene glycol. ^18,30 Alzet pumps (model 2001D for 24 h, model 1003D for 48 h, and model 2001 for 7 days; Alzet Corporation, Cupertino, CA) were primed overnight in saline with their openings facing upwards to prevent inflow of saline, and then filled with reagent. Intravenous catheters were custom made from polyurethane intravascular tubing (1.2 mm outer diameter, 0.69 mm inner diameter; cat # PY2 72-4431; Harvard Apparatus, Holliston, MA) pulled over an open flame to a 100 μm diameter over 3 mm and then gas-sterilized. They were also filled and connected to the pumps through their flow moderators, which were inserted all but 2 mm into the pump. Immediately after the SCI, the right jugular vein was exposed by blunt dissection after a skin incision in the anterolateral neck area. A subcutaneous tunnel was made by blunt dissection to the posterior-lateral cervical and posterior thoracic area for placement of the pump that was attached to the thick end of the catheter. Two ligatures were placed around the mid-cervical portion of the jugular vein. The more rostral ligature was gently retracted to stop blood flow while the caudal one was tightened, but leaving the vessel lumen open. A small (0.5 mm) hole was made in between two ligatures with microscissors, and the catheter tip was inserted into the vein to a 10–12 mm depth. The caudal ligature was tightened around the vein and catheter. Next, the flow moderator was inserted all the way into the pump to expel any potential blood from the catheter tip. The rostral ligature was tightened and secured the catheter to adjacent soft tissues. The wounds were sutured in layers. The patency of the infusions was confirmed by checking that the catheter tip was positioned correctly within the jugular vein lumen and by checking that almost no solution remained within the Alzet pump after the rats were euthanized.

Functional assessments

All animals in the 4 week experiment were tested for overground locomotor performance at weekly intervals. To reduce stress and inter-animal variability, they were handled over a period of 3 days and allowed to adapt to the test devices before testing. Open field locomotor function was performed using the Basso–Beattie–Breshnahan (BBB) scale. ³¹ Once a rat walked continuously in the open field (circular tub), two certified examiners scored the performance for 4 min. Baseline values were obtained on the day before surgery. Animals were tested on the 2nd day to ensure equal injury severities between groups and weekly, starting at 7 days post-injury. The rats were also tested in a sensorimotor grid walk test, which is particularly sensitive to dorsal column injury. ²⁰ Rats walked voluntarily on a 114×114 cm grid with 3.8 cm holes. The number of hindlimb footfalls were counted by two observers from different sides and recorded by a third person. The test was terminated after total 90 sec of walking was observed, and the total time of the test was recorded. Baseline values were determined on the day before injury.

Histological procedures

Depending upon the experiment, animals survived between 20 min and 4 weeks after the spinal cord contusion. Twenty minutes prior to processing for histology, the left jugular vein was exposed and 200 μL containing 200 μg rhodamine-conjugated LEA (Vector Laboratories, Burlingame, CA) was injected over 1 min. After 20 min, the rats were perfused with 200 mL ice-cold 0.1 M PBS followed by 200 mL 4% paraformaldehyde (PFA; Cat #19210; Sigma, St. Louis, MO) in phosphate buffer. The block of thoracic spine was excised and post-fixed overnight in PFA before dissection of the spinal cord, which was affixed to a strip of dental wax to straighten it, and cryoprotected in 30% sucrose overnight. A segment of 11 mm containing the injury epicenter in the center was collected. Multiple spinal cords were aligned and a reference tissue to orient each batch were cast in a single block of Tissue Freezing Medium (TMF-5, Triangle Biomedical Sciences Inc., Durham, NC) and frozen at −20°C. Every other 20 μm transverse section was collected on a cryostat onto SuperFrost plus glass slides (VWR, Radnor, PA) to produce five series with each section separated by 200 μm.

Before staining, slides containing the transverse sections were warmed and dried at 37°C for 60 min, and the freezing medium was removed. All other steps were at room temperature. Sections were incubated flat, and a wall was drawn with a slide marker (RPI, Mount Prospect, IL) around the edges of the slide to retain fluids. The slides were washed with PBS 3 times for 10 min in between all steps. To visualize LEA, every fifth slide was processed for immunohistochemistry to amplify the LEA signal (1:500; goat anti-rhodamine immunoglobulin G [IgG]; Cat SP-0602; Vector Laboratories; Burlingame, CA) using Alexa594-conjugated donkey anti-goat secondary antibodies (1:200; Cat#A11058; Invitrogen, Grand Island, NY). The same sections were immunostained for the endothelial marker RECA1 (1:1000; mouse anti-rat monoclonal antibody; catalog #MCA970; Serotec, Raleigh, NC), combined with Alexa488-conjugated donkey anti-mouse IgG as secondary antibody (1:200; Cat#A21202; Invitrogen). Oligodendrocytes were identified with mouse anti-APC antibody; 1:1000 (Cat # OP80, Millipore, Billerica, MA), with secondary Alexa488-conjugated donkey anti-mouse IgG as secondary antibody (1:200; Cat#A21202; Invitrogen). The slides were cover-slipped with fluoromount-G (Cat# 0100-01; Southern Biotech, Birmingham, AL).

Sections adjacent to the LEA-stained sections were used to document the extent of hemorrhage using the diaminobenzidine (DAB) staining step in which endogenous peroxidases in red blood cells convert the DAB substrate. ³² Briefly, the slides were washed with PBS twice for 10 min, and then for 10 min in 0.05 M Tris buffered Hanks saline (TBH) without sodium chloride. The DAB solution was prepared fresh and contained 0.4 mg/mL DAB (Cat #D5637, Sigma, St. Louis, MO), 0.6 mg/mL ammonium nickel sulfate and 0.6 μl/mL of 30% hydrogen peroxide, all in 0.05 M TBH. Slides were incubated in DAB solution for 5 min, rinsed three times for 10 min with PBS, washed in 0.1 M PB, then serially dehydrated up to 100% ethanol followed by xylene, and then cover-slipped in Permount (SP15-500, Fisher Scientific, Pittsburg, PA).

White matter was stained with an Eriochrome Cyanine (EC) staining protocol. ³³ Dried slides were immersed in xylene twice for 30 min to remove freezing medium remnants, and the sections were hydrated through a graded ethanol range up to 50%. EC solution consisted of 2 mL of 10% FeCl₃ (cat # 157740; Sigma) and 40 mL of 0.2% EC (cat # 32752; Sigma) in 0.5% aqueous H₂SO₄ (cat # 339741; Sigma) and brought to a final volume of 50 mL with ddH₂O). EC was applied to the slides for 10 min. The final step consisted of rinsing with ddH₂O and differentiating with 0.5% aqueous NH₄OH (cat # 221228; Sigma) for 30 sec. After termination of the reaction with a ddH₂O wash, slides were dehydrated and cover-slipped in Permount.

Adjacent sections were stained with cresyl violet (CV) to detect neuronal cell bodies. First, to clear cords from hemorrhage, slides were soaked to 3 min to 0.3% H₂0₂ solution (cat # HX0635-1; EMD Chemicals; Gibbstown, NJ). Next, sections were de-fatted with graded ethanol range to 95% and rehydrated back to 50% and ddH₂O and then placed in a 0.1% cresyl violet solution (in ddH₂0; cat # C1791; Sigma) for 8 min. Sections were differentiated in 0.1% acetic acid (cat A0808; Sigma) in 95% ethanol for 30 sec, dehydrated through an ethanol range to xylene, and then cover-slipped in Permount.

Quantitative measurements and statistical analyses

Stitched (mosaic) fluorescent images of whole transverse sections were taken on a DM 6000 Leica upright microscope with a 20x objective and Surveyor software (Objective Imaging, Cambridge, UK) combined with an Oasis Automation Controller (Objective Imaging, Cambridge, UK) driving the motorized stage. All images within a type of staining were taken with the same exposure time.

LEA+ or RECA+ microvessels intersecting grid lines were counted at a high magnification. Briefly, in Adobe Photoshop CS5 software (Adobe systems, San Jose, CA), a 800×800 μm grid with a 100 μm cell size was placed over the dorsal central area between dorsal horns and over the ventral area on one side of the spinal cord using the central canal as a guide (Fig. 1I). ³⁴ Tube-like structures were visually identified, and those intersecting the horizontal or vertical lines were counted manually. For the acute phase experiments, the injury epicenter was defined by the three sections with the lowest number of LEA+ microvessels, as determined by counts in one series of sections (each 200 μm apart) through the 11 mm length of the collected thoracic spinal cord segment. This epicenter was used as a reference point for quantifying the extent of hemorrhage, and the number of motor neurons and oligodendrocytes in adjacent series of sections. The penumbra was defined as the three sections 1 mm rostral and the three sections caudal to the epicenter. The average number of microvessels in the three sections through the epicenter, as well as the three sections in the rostral and caudal penumbrae, respectively, were calculated for each animal. ²¹ The average of both penumbra values for each rat were used to represent the penumbra value.

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FIG. 1.

Loss of tissue perfusion is rapid following spinal cord contusion. (A–H) are from a ventrolateral region of ventral horn (asterisk) and adjacent white matter (see darker area in I) in transverse sections 1 mm from the injury epicenter. (A–D) LEA staining. (E–H) RECA staining. A similar pattern of LEA- and RECA-stained microvessels is seen in gray and white matter of sham-operated rats (A,E). (B,F) Twenty minutes after the injury, the ventral horn is distorted and the number of vascular structures is decreased. (C,G) At 6 h, more vascular structures are detectable. (D,H) At 48 h post-injury, the gray matter is distorted and loss of structural integrity is evident. Note the patchy pattern of LEA- and RECA-stained vessels displaying vascular disintegration. (I) An Eriochrome Cyanine (EC)-stained transverse section through the normal thoracic cord showing the location of the counting grids used to quantify the microvasculature. (J–L) Shows the hemorrhagic area in transverse sections area at 20 min, 6 h, and 48 h post-injury. Bleeding is predominantly seen in the entire gray matter with small linear patches in the white matter. Scale bar in E=100 μm for A–H and in J=200 μm for I–L.

For quantification of the hemorrhagic area, gray scale images of DAB-stained sections were taken with a 10x objective and stitched with the abovementioned microscope hardware and software. Quantification of the hemorrhage area was performed using ImageJ 1.45s software (Wayne Rasband, NIH, Bethesda, MD). The image of the whole spinal cord was transformed to a binary image, and the percentage of black pixels representing the hemorrhagic area (DAB stained) was calculated. The numbers of the three sections at the epicenter were averaged, as well as those of the three sections at 1 mm rostral and 1 mm caudal to the penumbra, for every animal. The average of both penumbra values for each rat was used to represent the penumbra value.

Motor neurons were completely absent at the epicenter or at 1 mm from it. Therefore, the number of motor neurons in the ventral horn was counted manually in three cresyl violet- stained sections at 3 mm rostral from the epicenter, and recalculated as a percentage of sham-operated controls. Oligodendrocyte cell bodies were identified by APC staining and counted manually in the white matter of the right ventral quadrant of the cord in an adjacent series of three sections at 3 mm rostral from the epicenter. This number was also recalculated as a percentage of sham controls.

For the 4 week study, images of EC-stained sections of the cord were obtained as described. Quantification of preserved white matter was performed by calculation of EC- positive pixels in one series of sections (200 μm apart) through the collected 11 mm spinal cord segment. Briefly, after the transformation of each section to a binary image, the percentage of black pixels was calculated for every section, and the average of three consecutive images was calculated. The three sections with the minimum area of EC staining were defined as the injury epicenter. The penumbra was defined as three sections 1 mm rostral and caudal to the epicenter.

In experiments with two groups, the Student t test was used to determine whether statistical differences existed. ANOVA with Tukey's multiple comparison's test was used to compare more than two groups. Results of the behavioral testing were analyzed by repeated measures ANOVA and by paired Student's test for each individual rat. A value of p<0.05 was considered statistically significant. Microsoft Excel 2010 (Microsoft Co., San Jose, CA) was used to collect primary data and to create tables and graphs. ANOVA was performed with Prism 5 (GraphPad, La Jolla, CA). Final versions of figures were created with Adobe Illustrator software (Adobe systems, San Jose, CA).

Temporal profile of microvascular perfusion in the acute stage of SCI

The distribution of the LEA+ and RECA+ microvessels in the spinal cord of sham- operated animals was the same, with a typical dense capillary network in the gray matter compared with white matter (Fig. 1A, E). At 20 min after the contusion, the injury epicenter showed signs of severe damage with almost no LEA+ microvessels in the dorsal spinal cord (not shown). In the penumbra (1 mm from the epicenter), the ventral horns had very few detectable microvessels remaining, with some present in the white matter rim (Fig. 1B). RECA staining showed a similar pattern (Fig. 1F). At 6 h, more RECA+ microvessels were seen, but not a recovery of LEA+ vessels (not shown). In the penumbra, partial recovery of LEA+ vessels was seen in both gray and white matter (Fig. 1C). However, the microvascular density was much lower compared with that in the sham-operated animals. At 48 h, LEA and RECA staining showed the disintegration of the microvascular network, more so at the epicenter (not shown) than in the penumbra (Fig. 1D,H).

The number of microvessels was determined in two regions (Fig. 1I) within the spinal cord sections, combined and expressed as a percentage of sham. At the injury epicenter, the number of LEA+ or RECA+ microvessels was reduced to 7±2% or 13±4%, respectively, at 20 min. (Fig. 2A). The number of LEA+ epicenter microvessels did not significantly change over 48 h, when only 3±1% were observed (Fig. 2A,B). At 3 h, the number of RECA+ microvessels increased up to 25±3% (p<0.05 vs. 1 h) and stayed constant up to 24 h, after which they declined to 9±3% by 48 h (p<0.05 vs. 24 h; Fig. 2A,B). The recovery of RECA may indicate protein downregulation immediately following injury, or issues with perfusion-fixation. The discrepancy between the low LEA+ and higher RECA+ (p<0.001 for 12 and 24 h; p<0.05 for 3, 6, and 48 h) in the epicenter suggests that most of the surviving epicenter vessels were not perfused.

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FIG. 2.

Temporal changes in microvessel labeling reveals hypoperfusion following spinal cord contusion. The number of microvessels was counted in the two regions shown in Figure 1I at the injury epicenter and (A,B) and penumbra (C,D). The data from A and C are also shown in B and D, but are presented separately to visualize all data during the acute injury phase. A rapid drop of LEA+ microvessels remains low at the epicenter (A,B), whereas the perfusion recovers partially in the penumbra (C,D). The difference between LEA and RECA as a general marker for blood vessels suggests that a subset of vessels is not being perfused. Data are shown as average plus SEM. Group sizes are five per time point of injury and eight sham operated animals. The horizontal broken lines represent the p values for LEA and the solid lines represent those for RECA. Differences between LEA and RECA are indicated by asterisks between the two data points. *p<0.05; **p<0.01; ***p<0.001 by ANOVA with Tukey's multiple comparison's test.

In the adjacent rostral and caudal penumbra areas 1 mm away from the injury, the number of LEA+ or RECA+ microvessels was reduced to ∼30% of sham at 20 min (Fig. 2C). This reduction persisted at 1 h, but recovered up to 55±5% (LEA) and 64±10% (RECA), respectively, at 6 h post-injury (p<0.001; 1 vs. 6 h). This recovery continued up to 24 h, when 40±4% (LEA) and 58±3% (RECA) were detected (Fig. 2C,D). At the 48 h time point, the microvessels were reduced to 23±1% (LEA) and 38±3% (RECA)(p<0.001 and p<0.05 vs. 6 h). The discrepancy between the LEA+ and RECA+ (p<0.01) microvessels from 12 to 48 h suggests that a proportion of them were not perfused.

Temporal profile of hemorrhage in the acute stage of SCI

DAB staining was used to document the dynamics of the hemorrhagic area. Spinal cords of sham-operated animals were negative for DAB staining (not shown). At 20 min post-injury, the hemorrhagic area was predominantly located in the gray matter, and had a compact appearance (Fig. 1J). At the epicenter, the hemorrhage completely occupied the gray matter with small patches in adjacent white matter. In the penumbra, the hemorrhage was located more centrally. By 6 h, the overall area was similar to that seen at 20 min (Fig. 1K). Compared with at the earlier post-injury times, the hemorrhagic areas appeared lighter at 6 h. At 48 h post-injury (Fig. 1L), the transformation of the hemorrhagic area seemed to continue. At the epicenter, the lighter colored hemorrhagic area expanded into the white matter, reaching the dorsal outer surface. The overall density of DAB decreased, with some areas oriented radially. These changes in the compactness of hemorrhage area may be attributed to edema, heme destruction, and infiltration of inflammatory cells, which would also be stained because of their endogenous peroxidases.

The area of hemorrhage was quantified. Already at 20 min post-injury, hemorrhage was maximal at the epicenter and penumbra 1 mm away, occupying 29±3% and 15±1%, respectively, of the spinal cord cross-sectional area (Fig. 3A). The values at the epicenter were higher than at the penumbra at most post-injury times (p<0.05). The area of hemorrhage at the epicenter was lower at 24 and 48 h than at 20 min (p<0.01) and 1 h (p<0.05) post-injury (Fig. 3B). At the penumbra, the values were lower at 24 and 48 h than at 12 h (Fig. 3B; p<0.01). This reduction might be attributed to the breakdown of heme. ^9,35

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FIG. 3.

Hemorrhage and peri-spinal cord hyperemia occurs rapidly following spinal cord contusion. (A,B) The area of diaminobenzidine (DAB) staining for hemorrhage in the epicenter and penumbra of injured cord were quantified. At 20 min, the hemorrhage area was already maximal, and it decreased by 24 h, perhaps because of hemolysis. Rats are the same as in Figure 2. (*p<0.05; **p<0.01; ***p<0.001). (C,D) Doppler readings over the dorsal surface of the spinal cord with the sensor located just rostral to the injury epicenter were performed on a different set of rats. Each rat served as its own control; with 0 h values taken from immediately before the injury, and with measurements repeated over 48 h. n=4 rats. There is a rapid increase at 1 min after the injury with stable high values up to 12 h and decreasing flow to ∼50% of normal at 48 h (***p<0.001).

Laser Doppler flowmetry in the acute stage of SCI

Values were calculated within individual rats compared with their individual baseline readings which were considered as 100%. Acutely after injury (1 min), we observed a rapid increase up to 174±13%, with a further increment up to 218±10% at 2 h (Fig. 3C). The flow declined to ∼50% between 12 and 30 (Fig. 3C,D). The increase of overall flow suggests that the reduction of microvascular perfusion because of a spasm of the superficial arterioles. ³⁶

Intravenous (i.v.) MgCl₂ treatment improves microvasculature perfusion after SCI

At 24 h after the T9 contusion injury, animals infused with saline had fewer LEA+ than RECA+ microvessels in the combined epicenter and penumbra (52±5% vs. 36±4% of sham; p<0.05; Fig. 4A). Continuous i.v. infusion with MgCl₂ during a 24 h period resulted in a higher number of LEA+ microvessels (57±5% vs. 36±4% of sham in vehicle- treated group; p<0.05; Fig. 4A). Also, with MgCl₂ treatment, the number of LEA+ microvessels was equal to the number of RECA+ ones, suggesting that all surviving microvessels were perfused. At the epicenter, more RECA+ microvessels were seen with MgCl₂ (52±4% vs. 44±2% with vehicle; p<0.05; data not shown).

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FIG. 4.

Intravenous (i.v.) MgCl₂ treatment increased microvascular perfusion without increasing hemorrhage following spinal cord injury. (A) Shown are the percentages of RECA+ or LEA+ microvessels in the epicenter plus penumbra at 24 and 48 h. As shown also in a separate experiment (Fig. 2), the control phosphate-buffered saline (PBS)-treated animals displayed hypoperfusion status (LEA lower than RECA). The vasodilatory effect of MgCl₂ is still seen at 48 h, but the total number of microvessels is less than at 24 h. (B) There is no change in the area of hemorrhage with the MgCl₂ treatment at 24 h post-injury. n=5 per group. *p<0.05.

At 48 h, animals infused with saline had fewer LEA+ than RECA+ microvessels (19±4% vs. 36±4% of sham; p<0.05; Fig. 4A). Continuous i.v. infusion over the same period with MgCl₂ had protective effects on microcirculatory flow, with increased numbers of LEA+ microvessels (30±4% vs. 19±4%; p<0.05; Fig. 4A). However, the number of RECA+ microvessels was higher than that of the LEA+ ones, suggesting that a subset of the vessels were not perfused at this time. At 48 h, the MgCl₂ treatment may have been protective for microvessels, as more RECA+ vessels were counted in the penumbra (59±7% vs. 42±3% with vehicle; p<0.05; data not shown).

Importantly, the increase in microcirculatory blood flow in response to the MgCl₂ treatment did not result in increased hemorrhage in epicenter or penumbra, as measured at 24 h (Fig. 4B).

MgCl2 treatment does not have neuroprotective effects

Motor neurons die through an excitotoxic mechanism following spinal cord contusion, and magnesium counteracts excitotoxicity in animal models of traumatic and hypoxic injury. ^{18,37 –39} We therefore assessed ventral horn motor neuron survival at 24 h after injury in cresyl violet-stained sections. In both vehicle- and MgCl₂-infused animals, there was a total absence of motor neurons in the epicenter and at 1 mm from it (the penumbra), and we could not, therefore, count them. Compared with sham-operated rats (Fig. 5A), both vehicle- (Fig. 5B) and MgCl₂-infused animals (Fig. 5C), showed some survival of neurons in the ventral horn at 3 mm rostral from the injury epicenter. The number of motor neurons was not different in the vehicle- and MgCl₂-treated animals, being 21±2% and 20±3% of sham, respectively (p>0.05). Moreover, there was also loss of APC-positive oligodendrocytes in the ventral white matter at 3 mm rostral from the epicenter Fig. 5D–I). In sections adjacent to those used for motor neuron counts, the percentage of APC-positive oligodendrocytes in the ventral white matter was not different, with 30±1% and 35±3% of sham seen in vehicle and MgCl₂ treated rats, respectively (p>0.05). Therefore, despite the improved blood flow, the continuous infusion of MgCl₂ did not have neuroprotective or toxic effects during the acute injury phase.

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FIG. 5.

MgCl₂ treatment does not protect motor neurons or oligodendrocytes after spinal cord injury. (A) A transverse section through the thoracic spinal cord of a sham-operated rat stained with cresyl violet clearly shows neuronal cell bodies, particularly the large somatic motor neurons of the ventral horn. The boxes in the inset indicate areas of ventral horn (left) and white matter (right) shown in A–C and D–F and which were used for quantification of neurons and oligodendrocytes. Scale bar=100 μm. (B) With intravenous infusion of phosphate-buffered saline (PBS), fewer neurons are seen at 24 h after the contusion at 3 mm from the injury epicenter. (C) MgCl₂-treated rats also have fewer neurons. (D) APC-positive oligodendrocytes are equally distributed in the ventral white matter as shown in a transverse section from a sham-operated rat. Scale bar=200 μm. (E) With intravenous infusion of PBS over 24 h following a contusive injury, fewer oligodendrocytes are seen at 3 mm from the injury epicenter. (F) MgCl₂-treated rats also have fewer oligodendrocytes. (G–I) Higher magnification images of D–F. Scale bar=10 μm.

We tested whether a 7-day i.v. treatment with MgCl₂ would have neuroprotective effects following the T9 contusion. Functional recovery, as measured by weekly BBB (Fig. 6A) and grid walk (Fig. 6B) tests, was not affected positively or negatively by the treatment, as the groups did not differ significantly. There was also no difference in white matter sparing at the epicenter (48±3% vs. 51±4% of total cord area; Fig. 6C–E). There also was no difference between the number of LEA+ and RECA+ microvessels in the injury epicenter and penumbra (Fig. 6F).

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FIG. 6.

Locomotor function, white matter sparing, and microvessel quantity is not affected by a 7 day intravenous (i.v.) MgCl₂ treatment following spinal cord injury. (A) The Basso–Beattie–Breshnahan (BBB) score for overground locomotor function is not different in the vehicle phosphate-buffered saline (PBS) and MgCl₂ treated groups over a 4 week period. (B) Grid walk performance is also not significantly different. (C,D) Examples of Eriochrome Cyanine (EC)-stained transverse sections at the epicenter in control PBS (C) and MgCl₂ (D) treated rats show the same extent of white matter sparing at 4 weeks as quantified in (E). (F) In the same rats, the average percentage of RECA- and LEA-stained microvessels was not different. n=6 for PBS and 5 for MgCl₂.