Spinal cord edema following freezing injury in the rat: Relationship between tissue water content and spinal cord blood flow

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Spinal Cord Edema Following Freezing Injury in the Rat : Relationship Between Tissue Water Content and Spinal Cord Blood Flow Rui Wang, M.D., Kazumasa Ehara, M .D., and Norihiko Tamaki, M .D. Department of Neurosurgery, Kobe University, Kobe, Japan

Wang R, Ehara K, Tamaki N . Spinal cord edema following freezing injury in the rat : Relationship between tissue water content and spinal cord blood flow . Surg Neurol 1993 ;39 :348-54 . A spinal cord edema model was developed in the rat by inflicting a freezing injury at -40°C for 3 minutes . Regional spinal cord blood flow, tissue water content, and histology were examined . White matter edema bad extended several segments by 8 hours after the injury. Tissue water content increased by 20.6% at 24 hours . Spinal cord blood flow in surrounding tissues decreased by more than 25% 4 hours after the injury . The progression of spinal cord edema following freezing injury appeared to be due to disruption of the blood-spinal cord barrier. KEY WORDS : Freezing injury ; Edema; Water content ; Spinal cord blood flow; Rat

Secondary injury to the central nervous system following the primary lesion may often exaggerate the initial injuries of various etiologies . In such cases, edema is one of the important elements of the secondary injury, along with ischemia and metabolic disturbances . However, there have been few studies of spinal cord edema, and numerous questions about the pathophysiology of spinal cord edema remain unanswered . Edema following cold injury to the brain has been widely investigated, because it is reproducible, and the primary lesion is well demarcated [15] . We have developed a rat spinal cord edema model using local freezing injury . Histopathologic changes, tissue water content, and integrity of the blood-spinal cord barrier were examined in this model, along with the regional spinal cord blood flow (SCBF) in tissue surrounding the injury .

Address reprint requests to: Kazumasa Ehara, M .D ., Department of Neurosurgery, Kobe University School of Medicine, 7-5-1 Kusunokicho, Chuo-ku, Kobe 650, Japan. Received July 21, 1992 ; accepted November 9, 1992 .

1993

by Elsevier Science Publishing Co., Inc .

Materials and Methods Animal Preparation for Freezing Injury Male Wistar rats weighing 250-300 g were used in this study. Animals were anesthetized with 10% chloral hydrate (200 mg/kg, i .p .), fixed in a stereotactic frame and placed on a heating pad to maintain body temperature within the physiologic range . A 2-cm midline incision was made along the back . The T-8 spinous process was resected, and a hole of 2 mm diameter was made in the arch of the lamina with a dental drill . The dura was then exposed but preserved intact . Animals were divided into freezing-injury and control groups . The equipment for local freezing consisted of a plastic syringe containing liquid nitrogen and a copper wire of 1 .5 mm diameter, as shown in Figure 1 . One end of the wire extended from the syringe and was applied gently to the surface of the dura for 3 minutes . Temperature was measured using a two-channel digital thermometer, PTW-100A (Unique Medical Co . Ltd ., Komae City, Tokyo, Japan) . The temperature of the wire tip was approximately -60 ° C . In the control group, saline at room temperature was used instead of liquid nitrogen in the apparatus during an identical surgical procedure .

Histologic Observation and Tracer Study of the Blood-Spinal Cord Barrier Three animals from each group were sacrificed with an overdose of 10% chloral hydrate at 1, 4, 8, 24, and 48 hours after freezing injury . After laminectomy, approximately 2 cm of spinal cord surrounding the cryolesion were quickly dissected . For pathologic examination, each spinal cord specimen was fixed in 10% formalin, embedded in paraffin, cut axially into serial 5- to 10-µm slices, and stained with hematoxylin-eosin . The extent of blood-spinal cord barrier disruption was visualized by intravenous injection of 2% Evans blue (3 ml/kg body weight) 30 minutes prior to sacrifice . 0090-3019/93/$6 .00



Surg Neurol 1993 ;39 :348-54

Experimental Spinal Cord Edema

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40-

liquid nitrogen plastic syringe

Figure 1 . The instrument used to produce freezing injury; it consists ofa plastic syringe that contains liquid nitrogen and an extended copper wire . The tip of the wire was placed gently on the surface of the dura .

1

20-

U

opper wire

4 v CL

E m IMeasurement of Specific Gravity and Tissue Water Content (TWC) Animals (n = 80) were grouped into control and the freezing-injury groups . Eight animals from each group were sacrificed at 1, 4, 8, 24, and 48 hours after injury as described above . The specific gravity of 30-mg wet weight tissue was determined by the Nelson method [18], and the change in tissue water content was then calculated as previously described [18] . Measurement of SCBF Rats were anesthetized, paralyzed with pancromium bromide, tracheostomized, placed on ventilators, and catheterized through the femoral artery for monitoring of mean arterial blood pressure, pH Pao 2 , and PaCO 2 . A partial laminectomy of 2 mm in diameter was performed two levels below the cryolesion at T-10 to measure the SCBF of surrounding tissue . A laser Doppler flowmeter (LDF) (ALF 21, Advance Co Ltd., Tokyo, Japan) was used to make serial measurements of SCBF using a laser at a wavelength of 780 nm and a probe tip of 1 mm in diameter. This procedure, its reliability, and a comparison to other methods have been described in a previous report [28} . Regional SCBF was measured preinjury, and at 1, 10, 20, and 30 minutes, and 1, 4, 8, 24, and 48 hours after surgery in control (five animals) and freezinginjury (eight animals) groups . SCBF was normalized by the following formula : Normalized SCBF (%) =

0-

m

Measured SCBF X 100 . Preinjury SCBF

Data Analysis All data are expressed as the mean ± standard deviation . The unpaired t test was used for statistical analysis, and the null hypothesis was rejected when p < 0 .05 . Results The temperature of the dorsal surface of the spinal cord at the lesion reaches a minimum of -41 .0°C ± 4 .6°C

-20-

-40-

-60 preinjury freezing 1 min 2 min 3 min 10 min

Time after freezing injury Figure 2 . Temperature time course at the dorsal surface of the spinal cord at the site of the cryolerion . Vertical bars represent the SD of three determinations .

at the end of freezing (Figure 2) . The temperature then recovered rapidly to 10 .8°C -t 6.3 °C within 1 minute after freezing . The temperature of the spinal cord two levels below the freezing probe remained above -6 °C. No significant differences appeared in any physiologic parameter, including blood pressure, pH, PaCO 2 and Pa02 , between the control and freezing-injury groups (Table 1) .

Histology and Extravasation of Evans Blue Serial histologic changes in spinal cord white matter 7 mm below the lesion are shown in micrographs in Figure

Table

1 . Sequential Changes in Spinal Cord Arterial Blood Gas and Mean Arterial Blood Pressure Following Freezing Injury Group

Paw, (mm Hg)

Control (n = 5) 1 h 38 .2 ± 2 .3 4h 37 .7 ± . 19 8 h 37 .6 ± 1 .9 24 h 36 .0 ? 1 .5 48 h 37 .6 '- 1 .4 Freezing injury (n = 8) 1 h 37 .0 ± 1 .3 4 h 35 .6''-2 .6 8 h 37 .2±1 .9 24 h 36.8 ! 1 .6 48 h 38 .1!2 .3

Pa02 (mm Hg)

MABP (mm Hg)

103 .9`-12 .8 112 .5! 12 .8 106.1 ! 14 .5 104.8 ± 8 .7 103 .6 ± 7 .6

104 .6± 7 .1 101 .7 *- 12 .6 96 .7 ± 5 .8 96 .0 ± 5.3 103 .3 ' 5 .8

110 .7 3 100 .1± 103 .1'106 .6 '105 .7±

106 .6 ! 99 .4'100 .0'98 .0 ± 107 .03

9 .5 4 .8 6 .6 8 .9 7 .8

Abbreviation: MABP, mean arterial blood pressure . Note: Values are mean '_ SD .

6 .5 8 .2 3 .1 5 .7 4 .5



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Figure 3 .

Serial histologic changes in the posterior funiculus two segments (7 mm) below the cryolesion (HOE, x 100) . (A) Control, (B) I hours, (C) 4 hours, (D) 8 hours . (E) 24 hours . and (F) 48 hours after the freezing injury .

3 . At 1 hour after freezing, only occasional microhemorrhages were observed near the cryolesion with no visible expansion of extracellular space in the white matter (Figure 3 B) . At 4 hours after freezing, limited necrosis was observed in the white matter, accompanied by an increase in the extracellular space among the nerve fibers of the white matter in the posterior and lateral funiculus (Figure 3 C) . The surrounding white matter at both the cryolesion and at the adjacent level had a spongy appearance . At 8-48 hours after the cold injury, necrosis at the core of the lesion became more evident . The white matter exhibited interstitial edema along the entire axial section (Figure 3 D, E, F) . In the posterior horn at the level of the cryolesion after 8 hours karyopyknosis, blanching of the nuclei, and cytoplasmic vacuole formation was observed in the neurons . The time course of these histologic changes is summarized in Figure 4 . No extravasation of Evans blue was observed in control animals except in the dura . At 1 hour after the cryolesion, faint blue staining was identified at the freez-

ing lesion. At 4 hours after the lesion, extravasation of the dye became prominent ; however, the area of staining remained within 4 mm of the necrotic focus . From 8 hours and on after the injury, the area of extravasation expanded gradually both caudally and rostrally, and had extended 12 mm caudal to the cryolesion at 48 hours . Changes of Specific Gravity and Tissue Water Content The specific gravity of the spinal cord in the control group remained constant between 1 .0405 and 1 .0407 during the entire experiment . In the freezing group (Figure 5), the specific gravity remained unaltered until I hour after the cryolesion . It then decreased significantly to 1 .0362 (p < 0 .01), while TWC had increased by 12% (p < 0 .001) at 4 hours (Figure 5) . At 24 hours, the specific gravity reached its nadir at 1 .0338 (p < 0 .001), and TWC had increased by 20 .6% . The specific gravity and TWC remained at these levels during the remainder of the 24-hour study period . Changes in Regional Spinal Cord Blood Flow At the site of the freezing injury in the control group, SCBF remained within 10% of pretreatment flow



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Experimental Spinal Cord Edema

1h

4h

8h

24h

351

48h

Freezing center Adjacent (5mm)

Figure 4 . Serial histological changes after freezing injury at the site of the cryolesion, and 5 mm and 7 mm caudal to the lesion .

Adjacent (7mm) Edematous zone

® Necrotic zone

throughout the experiments (Figure 6 A) . In experimental animals, SCBF decreased to 46% of preinjury flow 1 minute after freezing . SCBF then recovered substantially by 10 minutes after the cryolesion, but remained slightly lower than that in control animals, and declined

Figure 5 . The specific gravity (A) and changes in the calculated tissue

again to flows of about 70% of baseline at 1 hour . At 4 hours after the cryolesion, SCBF had decreased further to 53%n of baseline (p < 0 .001), and continued to decrease to less than 50% of baseline at 8 hours, and to 44 .5% (p < 0 .001) at 24 hours . At 48 hours, SCBF had not decreased further, but remained below 50% of baseline . Nearby regional SCBF at the level of the injury and

water content (B) by the Nelson method in control and freezing injury groups. The vertical bars represent the SD of eight determinations . *Statistically significant change from the time-matched control (p < 0 .001) .

Figure 6. Regional spinal cord blood flow after freezing injury at the site of the cryolesion (T8), (A) ; and in the adjacent spinal cord (T-10), (B) . Vertical bars represent the standard deviations for control eve animals) and freezing injury (eight animals) measurements . ; 120 03 110' = 100' O 90 • soa a 7060c s0 a ° 40 -

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in the two adjacent segments was not reduced in either control or freezing groups (Figure 6 B) . In the freezing injury group, SCBF at this adjacent level decreased markedly during freezing and was at 63% of the preinjury level 1 minute after injury . SCBF then became higher than the preinjury level 10 minutes after freezing, and then decreased gradually, but remained within 7% of preinjury flow during the first 30 minutes after the cryolesion . SCBF was decreased by 15% (p < 0 .01) from control at 1 hour, and by 29% (p < 0 .001) at 4 hours . It reached its nadir at 63% of preinjury flow (p < 0.001) at 8 hours, and remained below 70% of preinjury flow after 8 hours . Discussion

Freezing Injury of the Spinal Cord Freezing injury to nervous tissue has been investigated for many years [5] . The initial phase of the injury, characterized by swollen astrocyte is short, and is followed by a remarkable increase in interstitial fluid . The primary lesion is well demarcated in the brain, but extensive white matter edema occurs throughout the injured hemisphere. This edema is reproducible, and has, therefore, provided a good model for research on brain edema [2,13,15,20] . There have been, however, few reports of pathological changes caused by freezing injury to the spinal cord . Collins et al. [4] reported damage to myelinated fibers at -3°C, probably due to extracellular hypotonicity . From -8°C to -12°C, damage to all myelinated fibers, as well as to some glial cells, was observed, but the vessels remained intact . Neurons were destroyed below -15 °C [4] . These temperatures were higher than those reported for cold-injury edema to the brain . In our experimental protocol, higher freezing temperatures produced by an acetone-dry ice mixture instead of liquid nitrogen, also failed to produce any edema in the surrounding white matter of the spinal cord (unpublished data) . The intracellular freezing point has been reported to be -15 °C [171 . However, because of its low thermal conductivity [21), temperatures under -30°C have been usually used to induce edema in the brain [7,261 . Cryolesions at -30°C for 3 minutes have been shown to induce damage to capillary endothelial cells in the brain [2] . White matter tolerance to cryolesions has been reported to be much higher than that of gray matter in the brain [11] . Based on these data, we used -40 °C to produce cryolesions in the present study .

Pathophysiology of Cold-Injury Edema Our histologic observations demonstrated microhemorrhages as early as 1 hour after freezing injury,, suggesting

Wang et al

the presence of damage to the spinal vascular endothelial cells . Separation of the myelin and a spongy appearance due to fluid accumulation in the extracellular space were found 4 hours after cryoinjury in the surrounding white matter. From 8 to 24 hours after the cryolesion, the edema extended to several segments of the full thickness of the spinal cord (Figure 4) . The increase in tissue water calculated from the specific gravity correlated with the histologic extension of the edema, with a time course similar to that previously observed after cryoinjury in the brain [2,13] . The histologic findings, the time course of the increase of tissue water content, and the results of the Evans blue study of vascular integrity, all suggested that the generation of edema was mainly due to a breakdown of the blood-spinal cord barrier . Milhorat et al . [ 161 reported the extravasation of Evans blue from blood vessels following freezing injury in the rat spinal cord at -70°C for 5 seconds. The marker was observed to move centripedally through the parenchyma and enter the central canal within 1-5 minutes . However, the degree of extravasation of Evans blue in their data was not remarkable . Recently, several biochemical mediators have been reported to contribute to the edema following cryolesions to the brain . Bradykinin [15], polyamine [14,27], arachidonic acid 1151, and free radicals [3] have been proposed to increase the permeability of the blood-brain barrier, and thus accelerate the progression of brain edema. These chemical mediators are also found in experimental spinal cord trauma [25] . However, the pathophysiology and metabolic changes underlying spinal cord edema are still unknown .

Changes in Spinal Cord Blood Flow "C-Antipyrine autoradiography, the radioactive microsphere method, and hydrogen clearance have been all used in the literature to measure spinal cord blood flow [25] . Recently, the laser Doppler flowmeter has been used in clinical and experimental research because it is less invasive and is especially useful for continuous monitoring [8,22,24] . A linear relationship has been demonstrated between the hydrogen clearance and LDF methods from 10-90 ml/100 glmin in normal rat spinal cord [28). Normalized SCBF, expressed as the percent change instead of the absolute value, should reduce the range of the variance among observations . A linear relationship between the percent change of SCBF measured by hydrogen clearance and LDF has been also reported previously [28) . There have been a few papers reporting changes in SCBF after cryolesions . Albin et al . [11 have reported that SCBF was unchanged in the dog spinal cord at 2 .5 hours after a 3 ° C cryolesion . Zielonka et



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[30] reported that SCBF was increased at 1 hour 10° -15 ° C trauma. Hansebout et al [10] demonstrated that SCBF was decreased by 50% after 3°C . Sakamoto and Monafo [23] reported 36% and 26% reductions of SCBF after 15°-18°C injuries . Although

Conclusions

after a

Freezing injury produced reproducible spinal cord

transient hyperemia was observed in the region adja-

course similar to the observed freezing injury increases

cent to the freezing site in that study, the reduction

of interstitial white matter edema .

edema in rats . White matter edema appeared to be due to a breakdown of the integrity of the blood-spinal cord barrier . Blood

flow decreased in the cord with a time

of SCBF remained predominant and persistent . However, the freezing temperatures used to produce injury in that study were quite different from those used in our experiments . Our results demonstrated that, after a cryolesion at

-40 °C, SCBF at the freezing site decreased markedly, recovered within 10 minutes, and then gradually decreased again . In adjacent areas, a transient increase in

flow was observed at 10 minutes, but SCBF was markedly reduced between 4 and 48 hours after injury . There have been several reports of changes in the blood

microcirculation after a cryolesion . Complete blood stasis at the cryolesion site was observed microscopically during cryoinjury, and circulation was then gradually reestablished . Transient hyperemia followed a few minutes later [2] . The time course of SCBF observed in our study was compatible with these disturbances of the microcirculation observed in brain cold injury . Disturbances of microcirculation and damage to vascular endothelial cells could be due to chemical mediators, such as noradrenaline

[19] or vasoactive amines

[29] . Recently, ischemia has been reported to be an important factor in secondary injury after acute spinal cord trauma [25] . In that study, a decrease in spinal cord microcirculation coupled with loss of local vascular

References . Albin MS, White Ill, Locke GE, Kretchmer HE . Spinal cord 1 hypothermia by localized perfusion cooling . (Letter) Nature (Lond)1966 ;210:1059-60 . 2 . Bakay L, Haque IU . Morphological and chemical studies in cerebral edema 1 . Cold induced edema . J Neuropathol Exp Neurol 1964 ;23 :393-418 . 3 . Chan PH, Longar SM, Fishman RA . Protective effects of liposome-entrapped superoxide dismutase on posttraumatic brain edema. Ann Neurol 1987 ;21 :540-7 . 4 . Collins GH, West NR, Parmely JD, Samson FM, Ward DA . The histopathology of freezing injury to the rat spinal cord . A light microscope study . 1 . Early degenerative changes . J Neuropathol Exp Neurol 1986;45 :721-41 . 5 . Denny-Brown D, Adams RD, Brenner C, Doherty MM . The pathology of injury to nerve induced by cold .J Neuropathol Exp Neurol 1945 ;4 :305-23 . 6 . Frei H), Wallenfang T, Poll W, Reulen HJ, Schubert R, Brock M . Regional cerebral blood flow and regional metabolism in cold induced oedema . Acta Neurochir (Wien) 1973 ;29 :15-28 . 7 . Go KG, Zijlstra WG, Flanderijn H, Zuiderveen F . Circulatory factors influencing exudation in cold-induced cerebral edema . Exp Neurol 1974 ;42 :332-8 . 8 . Haberl RL, Heizer ML, Ellis EF . Laser-Doppler assessment of brain microcirculation : Effect of local alterations . Am J Physiot 1989;256 (Heart Circ Physiot 25) :H1255-60 .

24 hours after

9 . HallenbeckJM, Leach DR, Durka AJ, Greenbaum I .J . The amount of circumscribed brain edema and the degree of post-ischemic neuronal recovery do not correlate well . Stroke 1982 ; 13 :797-804-

Our results demonstrated a similar time course for

10 . Hansebout RR, Lamont RN, Kamath MV . The effects of local cooling on canine spinal cord blood flow . Can J Neurol Sci 1985 ;12 :83-7 .

autoregulation caused a progressive reduction of SCBF in the initial few hours which persisted to injury .

decreases in SCBF and for changes in TWC . These changes were marked from

4 to 48 hours after the cry-

oinjury . Increases in local interstitial pressure have been hypothesized to reduce SCBF and disturb the microcirculation in the brain

[6] and spinal cord [20] . However,

even with decreased CBF and increased TWC in the brain, the remaining blood supply has been shown to provide sufficient nutrients for the neurons [12] . It has been also reported that there was no correlation between the extent of edema and the recovery of evoked potentials by the brain [9] . Thus, it remains to be shown whether spinal cord edema itself has effects on blood

flow or cord function . In our experiment the level of

11 . Hoffman WE, Albrecht RF, Miletich DJ. Regional cerebral blood flow changes during hypothermia . Cryobiology 1982 ;19 :640-5 . 12 . Katzman R, Clasen R, Klatzo I, Meyer JS, Pappius HM, Waltz AG . Report of joint committee for stroke resources . IV. Brain edema in stroke. Stroke t977 ;8 :512-40 . 13 . Klatzo I, Piraux A, Laskowski EJ . The relationship between edema, blood-brain barrier and tissue elements in a local brain injury . J Neuropathol Exp Neurol 1958 ;17 :548-64 . 14 . Koenig H, Goldstone AD, Lu CY . Blood brain barrierbreakdown in brain edema following cold injury is mediated by microvascular polyamines . Biochem Biophys Res Common 1983 ;116 :1039-48 . 15 . Maier-Hauff K, Baethmann AJ, Lange M, Schurer L, Unterberg A . The kallikrein-kinin system as mediator in vasogenic brain edema. Part 2 : Studies on kinin formation in focal and perifocal brain tissue . J Neurosurg 1984 ;61 :97-106 .

crease markedly, perfusion pressure would likely de-

16 . Milhorat TH, Johnson RW, Johnson WD . Evidence of CSF flow in rostral direction through central canal of spinal cord in rats . In : Matsumoto S, Tamaki N, eds . Hydrocephalus . Tokyo : SpringerVerlag, 1991 :207-17 .

crease enough to cause ischemia .

17 . Nei T . Mechanism of freezing injury to erythrocyres : Effect of

SCBF remained above the threshold of ischemic damage . However, were spinal cord tissue pressure to in-



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initial cell concentration on the post-thaw hemolysis . Cryobiology 1981 ;18 :229-37 . 18 . Nelson SR, Manrz M-L, Maxwell JA . Use of specific gravity in the measurement of cerebral edema. J Appl Physiol 1971 ;30 :268-71 . 19 . Osterholm JL, Mathews GJ . Altered norepinephrine metabolism following experimental spinal cord injury . Part I : Relationship to hemorrhagic necrosis and postwounding neurological deficits . J Neurosurg 1972 ;336 :86-94 . 20 . Palleske H . Experimental investigations on the regulation of the spinal cord circulation III : The regulation of the blood flow in the spinal cord altered by oedema. Area Neurochir (Wien) 1969 ;21 :319-27 . 21 . Rowbotham GF, Haigh AL, Leslie WG . Cooling cannula for use in the treatment of cerebral neoplasms . Lancet 1959 ;7062 :12-15 . 22 . Rundquist I, Smith QR, Michel ME, Ask P, Oeberg PA, Rapoport SI . Sciatic nerve blood flow measured by laser Doppler flowmetry and "C-iodoantipyrine . Am J Physiol 1985 ;248 :H311-H7 . 23 . Sakamoro T, Monafo WW . Regional spinal cord blood flow during local cooling . Neurosurgery 1990;26:958-62 . 24 . Skarphedinsson JO, Handing H, Thoren P . Repeated measurements of the cerebral blood flow in rats . Comparison between

the hydrogen clearance method and Laser-Doppler flowmetry . Aces Physiol Stand 1988;134 :133-4225 . Tator CH, Fehlings MG . Review of secondary injury theory of acute spinal cord trauma with emphasis on vascular mechanisms . J Neurosurg 1991 ;75 :15-26 . 26 . Torack RM, Terry RD, Zimmerman HM . The fine structures of cerebral fluid accumulation . 1 . Swelling secondary to cold injury . Am J Pathol 1959 ;35 :1135-47 . 27 . Trout JJ, Koenig H, Goldstone AD, Lu CY . Blood-brain barrier breakdown by cold injury . Polyamine signals mediate acute stimulation of endocytosis, vesicular transport, and micro villus formation in rat cerebral capillaries. Lab Invest 1986 ;55 :622-31 . 28 . Wang R, Ehara K, Fujita K, Tamaki N, Matsumoto S . Blood flow, C02 response and autoregulation in the rat spinal cord by LaserDoppler flowmetry and hydrogen clearance . Brain Nerve (Tokyo) 1991 ;43 :649-55 . 29 . Wieloch T. Neurochemical correlates to selective neuronal vulnerability . Prog Brain Res 1985 ;63 :69-85 . 30 . Zielonka JS, Wagner FC Jr, Dohrmann GJ . Alterations in spinal cord blood flow during local hypothermia . Surgical Forum . 1974 ;25 :434-6.