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Journal of Cerebral Blood Flow & Metabolism logoLink to Journal of Cerebral Blood Flow & Metabolism
. 2010 Dec 15;31(4):1143–1154. doi: 10.1038/jcbfm.2010.208

Combinational therapy using hypothermia and the immunophilin ligand FK506 to target altered pial arteriolar reactivity, axonal damage, and blood–brain barrier dysfunction after traumatic brain injury in rat

Yasutaka Oda 1,2, Guoyi Gao 1,3, Enoch P Wei 1, John T Povlishock 1,*
PMCID: PMC3070975  PMID: 21157473

Abstract

This study evaluated the utility of combinational therapy, coupling delayed posttraumatic hypothermia with delayed FK506 administration, on altered cerebral vascular reactivity, axonal injury, and blood–brain barrier (BBB) disruption seen following traumatic brain injury (TBI). Animals were injured, subjected to various combinations of hypothermic/FK506 intervention, and equipped with cranial windows to assess pial vascular reactivity to acetylcholine. Animals were then processed with antibodies to the amyloid precursor protein and immunoglobulin G to assess axonal injury and BBB disruption, respectively. Animals were assigned to five groups: (1) sham injury plus delayed FK506, (2) TBI, (3) TBI plus delayed hypothermia, (4) TBI plus delayed FK506, and (5) TBI plus delayed hypothermia with FK506. Sham injury plus FK506 had no impact on vascular reactivity, axonal injury, or BBB disruption. Traumatic brain injury induced dramatic axonal injury and altered pial vascular reactivity, while triggering local BBB disruption. Delayed hypothermia or FK506 after TBI provided limited protection. However, TBI with combinational therapy achieved significantly enhanced vascular and axonal protection, with no BBB protection. This study shows the benefits of combinational therapy, using posttraumatic hypothermia with FK506 to attenuate important features of TBI. This suggests that hypothermia not only protects but also extends the therapeutic window for improved FK506 efficacy.

Keywords: amyloid precursor protein, blood–brain barrier, brain arteriolar reactivity, diffuse axonal injury, hypothermia, immunosuppressant

Introduction

Traumatic brain injury (TBI) remains a major health-care problem with devastating societal costs. Despite significant progress in our understanding of TBI, its management has remained elusive (Narayan et al, 2002; Marklund et al, 2006; Dietrich et al, 2009). To date, many therapeutic approach agents have been brought to clinical trial, yet they have not proved efficacious (Narayan et al, 2002; Roberts et al, 2004). Recently, there has been interest in the use of posttraumatic hypothermia, with supportive data emerging from some clinical groups, whereas others have reported less compelling findings (Jiang, 2009; Hutchison et al, 2008; Peterson et al, 2008). Although most have framed the utility of experimental hypothermia in terms of global neuroprotection, involving reduced brain metabolism (Bacher et al, 1998), attenuation of radical-mediated production (Globus et al, 1995), and/or attenuation of traumatically induced brain edema and elevated intracranial pressure (Kawai et al, 2000), experimental studies have suggested that hypothermic intervention may offer an added benefit by elongating the therapeutic window over which other previously ineffective therapies can now provide protection (Dietrich et al, 2009; Povlishock and Wei, 2009).

We have recently shown in rats that the use of delayed, posttraumatic hypothermia provides partial cerebrovascular protection (Suehiro et al, 2003; Ueda et al, 2003). Importantly, this protection proved complete when delayed hypothermia was coupled with the use of radical scavengers at times at which they normally exerted no protective effects (Baranova et al, 2008). Realizing the limitations of one hypothermic study, with one agent and one end point, we now extend these studies, investigating the utility of hypothermia coupled with another agent, tacrolimus (FK506), which has been shown to be neuroprotective only early in a posttraumatic period (Singleton et al, 2001). FK506 has been reported to attenuate TBI-induced impaired axonal transport and axonal swelling (Singleton et al, 2001; Marmarou and Povlishock, 2006), as well as ischemic-induced neuronal cell death and infarct volume (Furuichi et al, 2003) when administered either before injury or early in the postinjury course. To explore the utility of a combinational approach coupling hypothermia and FK506 in delayed temporal framework postinjury, we assessed the utility of these combined approaches in attenuating not only concomitant axonal damage but also microvascular dysfunction and focal blood–brain barrier (BBB) dysfunction, all of which are major features of human TBI (Povlishock and Katz, 2005).

Materials and methods

General Preparation

All experimental procedures used protocols approved by the Institutional Animal Care and Use Committee at the Virginia Commonwealth University. A total of 28 adult male Sprague–Dawley rats, weighing 360 to 492 g (435±12 g, mean±s.e.m.), were used. Animals were housed in individual cages on a 12-hour light–dark cycle, with free access to water and food. Animals were anesthetized intraperitoneally with sodium pentobarbital (60 mg/kg). The femoral artery was cannulated with a PE50 catheter (Becton Dickinson, Sparks, MD, USA) for monitoring arterial blood pressure (PowerLab, AD Instruments, Colorado Springs, CO, USA) and blood sample collection for determining arterial oxygen tension (PaO2), arterial carbon dioxide pressure (PaCO2), and pH values (Stat Profile pHOx, Nova Biomedical, Waltham, MA, USA). Blood gas samples were analyzed using an alpha-stat management protocol in which the blood gas samples were measured at 37°C regardless of the animal's body temperature. These samples (100 μL) were collected 10 minutes before injury, and at 1, 2, 4, 5, and 6 hours after injury. The femoral vein was cannulated with a PE50 catheter for pharmacological agent administration and FK506/vehicle. After tracheotomy, animals were ventilated (Harvard Apparatus, Holliston, MA, USA). Pancuronium bromide (3 mg/kg) was administrated intravenously following lateral fluid percussion injury (LFPI) to produce skeletal muscle paralysis. The resting PaCO2 was maintained between 35 and 40 mm Hg, by adjusting the rate and/or volume of the respirator. Body temperature was maintained at 37°C using a heat lamp and/or a heating pad throughout the experiment, except during hypothermia.

The Induction of Hypothermia and FK506 Delivery

Hypothermia was accomplished by whole body cooling. Body and brain temperatures were measured using a rectal thermometer (Yellow Spring Instruments, Yellow Spring, OH, USA) and a thermistor in the temporalis muscle (Physitemp Instruments, Clifton, NJ, USA), respectively. The temporalis muscle temperature was used because it parallels brain temperature. Typically, 13 to 15 minutes of ice bag use was required to achieve the target rectal temperature of 33°C. Hypothermia was initiated 1 hour postinjury and maintained at 33°C for 60 minutes, followed by rewarming over a span of 90 minutes. Rewarming was accomplished using a protocol detailed previously (Suehiro et al, 2003). Animals also received either FK506 (Astellas Pharma US, Deerfield, IL, USA) or vehicle delivered intravenously 90 minutes postinjury. A single 3 mg/kg of FK506 in 0.9% sterile saline to a total volume of 1.0 mL was administrated over a 20-minute period. This dosage was based on previous work from our laboratory (Marmarou and Povlishock, 2006). The vehicle (0.9% sterile saline) was administrated using the same protocol. In these studies, we arbitrarily implemented the above-identified protocols.

Experimental Traumatic Brain Injury

Rats were subjected to moderate LFPI or a sham injury. The LFPI model was the same as described previously (Wei et al, 2009). In brief, rats were placed in a stereotaxic frame, and a midline sagittal incision exposed the skull. A 4.8-mm circular craniotomy was prepared midway between the bregma and the lambda on the left side of the skull. Two 3/16-inch long screws (Small Parts, Logansport, IN, USA) for fixation were inserted into 1-mm holes drilled into the left frontal and occipital bones. The top portion of the Leur-Loc hub (Becton Dickinson, Franklin Lakes, NJ, USA), cut away from the 20-G needle, was positioned over the dura mater and rigidly affixed to the two screws using dental acrylic (Hygenic Corporation, Akron, OH, USA). After the dental acrylic hardened, rats were disconnected from the respirator and rapidly connected to the LFPI device (Custom Design and Fabrication, Richmond, VA, USA) through a spacing tube. Rats were injured at 2.0 atmospheres (range: 1.91 to 2.03 atmospheres), consistent with a moderate TBI. The pressure pulse measured by the transducer was displayed on a storage oscilloscope (Tektronix, Beaverton, OR, USA), and the peak pressure was recorded. After injury, all animals were promptly ventilated with room air and reconnected to the blood pressure monitoring device. The surgical procedures for sham injury animals were the same as described above.

Visualization and Assessment of the Cerebral Microcirculation

After injury, the assembly hub, screws, and dental acrylic were removed en bloc. A 2 × 4 mm2 rectangular craniotomy was made over the right parietal bone on the contralateral side and the dura mater was cut. The dura mater on the side ipsilateral to the injury was then exposed and cut. Next, a cranial window was installed over the brain surface to encompass both craniotomies, and fixed in place by bone wax and dental acrylic. As described previously (Levasseur et al, 1975), the cranial window consisted of a stainless steel ring with three outlets. Two of the outlets served as inflow and outflow paths for the perfusion and clearance of selected vasoactive agents, while the free end of the other outlet was set at a predetermined height to achieve an ICP of 5 mm Hg. The space under the cranial window and the three outlets was filled with sterile artificial cerebrospinal fluid, with the pH adjusted to 7.35 by equilibration with a 6% O2 and 6% CO2 gas mixture balanced with N2. Pial microcirculation was visualized and pial arteriolar diameters were measured using a Vickers image-splitting device (Vickers Instruments, Maiden, MA, USA). Typically, a minimum of four arteriolar segments in each site ipsilateral and contralateral to the injury were evaluated. The vasodilator acetylcholine (ACh) was used in two different concentrations to assess vascular dilation after its application in the cranial window. Acetylcholine (Sigma, St Louis, MO, USA), well known to elicit endothelial-dependent vasodilatation (Kontos et al, 1988), was dissolved in artificial cerebrospinal fluid to achieve final concentrations of 10−7 and 10−5 mol/L and then applied through the cranial window. After application, ACh was allowed to remain in place for 2 to 4 minutes. Vascular reactivity to ACh was expressed as percentage change from the baseline diameter at each measurement time.

Experimental Design

In this study, vascular function, the burden of axonal damage, and BBB dysfunction were assessed after TBI, followed by hypothermia, the administration of FK506, or the combination thereof. Animals were arbitrarily divided into five groups. Each group contained five animals.

Group 1: Sham Surgery and the Administration of FK506

These animals underwent the same surgical procedures used in all other animals. However, they were not subjected to LFPI. Vascular reactivity to the two concentrations of ACh was assessed 90 minutes before FK506 administration, and again at 4, 5, and 6 hours after the first measurement of vascular reactivity (Figure 1).

Figure 1.

Figure 1

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This chart shows the time course for each experimental group. LFPI, lateral fluid percussion injury.

Group 2: Lateral Fluid Percussion Injury with no Treatment

These animals were subjected to LFPI and administered the vehicle 90 minutes postinjury. Body temperature was maintained at normothermic levels. Vascular reactivity to the two concentrations of ACh was assessed at 4, 5, and 6 hours after injury (Figure 1).

Group 3: Lateral Fluid Percussion Injury Followed by Hypothermia

These animals were subjected to LFPI and 1 hour postinjury, and were cooled to 33°C. Hypothermia was then maintained for 60 minutes. After vehicle delivery 90 minutes postinjury, animals were rewarmed to normothermic levels over an additional 90-minute period. Thereafter, vascular reactivity to ACh was evaluated at 4, 5, and 6 hours postinjury (Figure 1).

Group 4: Lateral Fluid Percussion Injury Followed by Delayed FK506 Administration

These animals were injured and FK506 was injected 90 minutes postinjury. Vascular responses to ACh were assessed at 4, 5, and 6 hours postinjury (Figure 1).

Group 5: Lateral Fluid Percussion Injury Followed by Hypothermia and FK506 Administration

At 1 hour postinjury, a 60-minute period of hypothermia was induced, followed by a 90-minute period of rewarming. These animals also received FK506 administration 90 minutes postinjury, with vascular reactivity to ACh assessed at 4, 5, and 6 hours postinjury (Figure 1).

In these vascular reactivity studies, investigator blinding was impossible to achieve because of the complexity of the experimental design and the fact that directly observed vasoactive responses strongly suggested a therapeutic versus nontherapeutic intervention.

Tissue Preparation

For evaluating the burden of axonal damage following TBI and hypothermia/FK506, we analyzed axonal damage in the corpus callosum, the deep cortical layers of the mediodorsal neocortex, and dorsolateral thalamus using strategies described previously (Gao et al, 2010). We used the same animals evaluated in groups 1 to 5 for the conduct of detailed axonal analyses. At 6 hours postinjury after the measurement of vascular reactivity to ACh (in groups 1 to 5), rats were killed with an overdose of euthanasia solution and transcardially perfused with 4% paraformaldehyde and 0.1% glutaralaldehyde in 0.1 mol/L Millonig's phosphate buffer. Each brain was coronally blocked between the optic chiasm and the midbrain to include the parietal and temporal cortices, hippocampus, and thalamus. For tissue sectioning, the brain blocks were flat mounted on a metal plate with cyanoacrylate. Blocks were coronally sectioned at 40 μm in 0.1 mol/L phosphate buffer using a vibratome (Leica Biosystems, St Louis, MO, USA). Coronal sections (n=60) were serially collected in alternating wells with each well containing adjacent sections, starting from 1,600 μm caudal to the anterior commissure. Systematic uniform sampling of coronal sections was initiated from a random starting well, with every fourth section collected for a total of 15 sections per animal. Additional sections were stored in Millonig's buffer in 12-well culture plates (Falcon, Newark, DE, USA) for use in the analysis of BBB dysfunction detailed below.

Immunocytochemistry for Axonal Damage and Blood–Brain Barrier Disruption

Sections were processed for visualization of an antibody targeting the amyloid precursor protein (APP), a marker of impaired axonal transport and axonal damage using a previously reported protocol (Stone et al, 1999). In brief, the sections were reacted with 0.3% H2O2 in phosphate-buffered saline (PBS) for 30 minutes to block endogenous peroxidase and microwaved in citric acid buffer while maintaining a 45°C maximum temperature for 5 minutes. The sections were then allowed to cool for 20 minutes. The sections were preincubated for 1 hour in 10% normal goat serum with 0.2% Triton X in PBS and then incubated for 18 hours with the rabbit anti-C-APP (Invitrogen, Carlsbad, CA, USA) diluted 1:1,000 in 1% normal goat serum in PBS. Next, the sections were incubated for 1 hour with biotinylated goat anti-rabbit immunoglobulin G (IgG) (Vector Laboratories Inc., Burlingame, CA, USA) diluted 1:1,000 in 1% normal goat serum in PBS. The reaction product was visualized by incubation for 1 hour in avidin-biotinylated enzyme complex (Vectastain ABC kit, Vector Laboratories Inc.), followed by 0.05% diaminobenzidene, 0.01% H2O2, and 0.3% imidazole in 0.1% mL sodium phosphate buffer for 15 minutes. The sections were mounted on 0.5% gelatin-coated glass slides, serially dehydrated, and coverslipped.

For IgG staining, a marker of BBB disruption (Ellison et al, 1990), other sections (15 sections per animal) were reacted with 0.3% H2O2 in PBS for 30 minutes. After blocking of endogenous peroxidase activity, the sections were preincubated for 1 hour in 10% normal rabbit serum with 0.2% Triton X in PBS and then incubated for 18 hours with the goat anti-rat IgG (Bethyl Laboratories, Montgomery, TX, USA) diluted 1:3,000 in 1% normal rabbit serum in PBS. The next day, the sections were incubated for 1 hour with biotinylated rabbit anti-goat IgG (Vector Laboratories Inc.) diluted 1:500 in 1% normal rabbit serum in PBS. Other steps were the same as for APP immunostaining.

Quantitative Analysis of Axonal Damage and Blood–Brain Barrier Disruption

After completion of APP immunocytochemical procedures, the slides were transferred in a blinded manner to an Eclipse 800 microscope (Nikon, Tokyo, Japan), interfaced using a computer-assisted imaging system DP Controller, version 3.2 (Olympus Corporation, Tokyo, Japan). Consistent regions of the corpus callosum, deep cortical layers of the mediodorsal neocortex, and dorsolateral thalamus were enlarged to a magnification of × 10 and saved as TIFF (tagged image file format) (Figures 2B to 2G). All loci were selected on the side ipsilateral to the injury. We used two different sampling areas in the corpus callosum, which were located at 1.5 and 2.5 mm from the midline (Figure 2A). On the basis of our previous experience (Gao et al, 2010), the image was viewed on a monitor using image analysis software, IPLab, version 3.7 (BD Biosciences Bioimaging, Rockville, MD, USA) and changed to gray scale. The APP-immunoreactive axonal profiles were outlined and overlaid with cyan color to suppress background immunoreactivity. The sampling area in the corpus callosum was delineated by a rectangle measuring 500 × 200 μm2 that was superimposed over the specified region. A rectangle measuring 500 × 500 μm2 was applied in the deep cortical layers of the mediodorsal neocortex and dorsolateral thalamus. The damaged APP-immunoreactive axonal profiles within these rectangles were then counted. This number was expressed as the density of damaged axons per unit area. For the corpus callosum and deep cortical layers of the mediodorsal neocortex, 15 alternate serial sections from the same tissue block were analyzed in this manner, whereas 12 sections were analyzed in the dorsolateral thalamus. These sections were randomly selected to prevent bias in their analysis.

Figure 2.

Figure 2

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Illustration and images of those regions sampled to assess the burden of axonal damage and altered blood–brain barrier status. (A) The sampling areas used to assess axonal damage are delineated by a rectangle in the corpus callosum and by a square in the deep cortical layers of the mediodorsal neocortex or the dorsolateral thalamus. The photomicrographs of the representative section of the rat brain in group 2 show numerous damaged APP-immunoreactive axons within the corpus callosum at 2.5 mm from the midline (B), deep cortical layers of the mediodorsal neocortex (D), and the dorsolateral thalamus (F). In contrast, in group 5, the numbers of APP-immunoreactive axonal profiles within the corpus callosum at 2.5 mm from the midline (C), deep cortical layers of the mediodorsal neocortex (E), and dorsolateral thalamus (G) are markedly reduced. Note that the area of IgG immunostaining in group 5 (I) expands on the side contralateral to the injury, and is larger than that seen in group 2 (H). Scale bars (panels B to G)=100 μm.

For quantitative analysis of BBB disruption, the section containing IgG immunostaining was saved as TIFF at a magnification of × 0.5 using the Eclipse 800 microscope (Nikon) interfaced with a computer-assisted imaging system SPOT software, version 4.7 (Diagnostic Instruments, Sterling Heights, MI, USA) (Figures 2H and 2I). Using the same approach used for APP counting, the area of IgG immunostaining was outlined and overlaid with cyan color and measured within a rectangle measuring 1,500 × 1,100 μm2 that included each section. Overall, 15 alternate serial sections from the same tissue block were analyzed in this manner.

Statistical Analysis

Statistical analysis was performed using the statistical software SPSS, version 16.0 (SPSS Inc., Chicago, IL, USA). All data were presented as mean±s.e.m. The physiologic parameters, laboratory data, and vascular reactivity to ACh, which were normally distributed, were analyzed by one-way ANOVA (analysis of variance). When a significant difference was found, multiple comparisons among time points in the same group and groups at each time point were performed using Bonferroni's test and Scheffé's test, respectively. The comparison between contralateral and ipsilateral vascular reactivity at each time point was analyzed using the paired T-test corrected by the Bonferroni method. The number of damaged axons and the area of IgG staining, which were not normally distributed, were analyzed by the Kruskal–Wallis test, followed by the Bonferroni test for multiple comparisons. A value of P<0.05 was considered to be statistically significant.

Results

Physiologic Observations

Other than the sham group, a total of 20 rats were subjected to LFPI and evaluated fully with the exception of group 2 (vida infra). Owing to rigorous exclusion criterion, two animals in group 3 and one animal in group 4 were excluded because of airway disconnection and/or cranial window failure. They were replaced with animals managed and evaluated in a consistent manner. As detailed below, group 2 witnessed consistent animal failure owing to the adverse consequences of TBI without any form of treatment.

There were no significant differences in baseline body weight and hematocrit between groups. The rectal temperature in the normothermic groups (groups 1, 2, and 4) was maintained at 37°C over the entire experimental period (Figure 3A). The temporalis muscle temperature was ∼0.5°C to 1.0°C lower than the rectal temperature (Figure 3B). In the hypothermic groups (groups 3 and 5), rats were cooled to a brain temperature of 32°C within 15 minutes (Figure 3B). No significant differences were found in the intervals required to reach this temperature between groups 3 and 5 (12.8±0.9 minutes and 14.6±0.7 minutes, respectively). Rats were maintained at this level for 60 minutes and were then rewarmed to normothermic levels over a span of 90 minutes. There were no significant differences in rewarming rates between groups 3 and 5. Table 1 shows the time course measurements of the mean arterial blood pressure and blood gas analyses. The mean values of PaO2 during the hypothermic period (2 hours postinjury) in groups 3 and 5 were significantly higher than those in groups 1 and 2 and those in group 2, respectively, and tended to be higher than those in other measurement time points in the same group (Table 1). Other than these physiologic changes, all other physiologic variables were within normal physiologic limits.

Figure 3.

Figure 3

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This graph illustrates the changes of the mean rectal and mean temporalis muscle temperatures throughout the duration of this study. The data points for (A) rectal and (B) temporalis temperatures represent 5-minute intervals. Values are expressed as mean±s.e.m.

Table 1. Physiologic parameters.

Variables Group Measurement period
    Preinjury 1 hour 2 hours 4 hours 5 hours 6 hours
  1 106±3 107±3 103±2 108±2 110±4 104±4
  2 104±3 102±3 102±3 101±2 95±5 97±5
MABP (mm Hg) 3 107±3 100±2 97±3 104±2 100±3 100±2
  4 106±2 106±1 103±1 103±3 98±5 100±7
  5 107±3 103±1 100±2 97±3 99±4 100±3
               
  1 7.40±0.02 7.41±0.03 7.42±0.02 7.42±0.02 7.41±0.02 7.41±0.02
  2 7.39±0.01 7.41±0.01 7.41±0.01 7.41±0.01 7.41±0.01 7.39±0.01
pH 3 7.41±0.01 7.42±0.01 7.40±0.02 7.40±0.01 7.40±0.01 7.40±0.01
  4 7.40±0.03 7.42±0.02 7.41±0.03 7.41±0.03 7.41±0.03 7.41±0.02
  5 7.43±0.01 7.45±0.01 7.42±0.02 7.45±0.02 7.43±0.02 7.43±0.01
               
  1 85±3 87±3 84±4 85±4 81±5 85±5
  2 84±4 85±5 82±4 85±3 83±4 83±3
PaO2 (mm Hg) 3 87±5 87±3 107±6a 78±3 82±6 81±6
  4 93±8 89±4 87±4 87±4 86±3 87±3
  5 80±2 81±3 105±5b 83±2 80±4 84±2
               
  1 40±2 36±2 37±2 36±1 37±1 36±1
  2 38±1 38±1 38±1 36±1 35±1 36±1
PaCO2 (mm Hg) 3 40±1 37±1 37±2 38±1 36±1 37±1
  4 41±2 38±2 38±2 39±2 39±2 39±2
  5 39±2 38±1 38±1 37±2 38±2 36±2
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MABP, mean arterial blood pressure; PaCO2, arterial carbon dioxide pressure; PaO2, arterial oxygen tension.

Values are expressed as the mean±s.e.m.

a

Significant difference compared with the values of groups 1 and 2 at same measurement point (P<0.05).

b

Significant difference compared with the value of group 2 at same measurement point (P<0.05).

Brain Arteriolar Reactivity after Sham Injury and Lateral Fluid Percussion Injury

Vascular reactivity in sham-injured animals (group 1) was measured on the side contralateral or ipsilateral to injury. In Figures 4 and 5, vascular reactivity to ACh at 10−7 mol/L and at 10−5 mol/L on the contralateral side was the same as that obtained for the ipsilateral side. In group 1, preinjury resting arteriolar diameters equaled 42±2.4 μm mean±s.e.m., with the vasodilator responses to ACh at 10−7 mol/L and at 10−5 mol/L revealing an increase in the diameter of 9.5%±0.6% and 19.0%±0.7%, respectively (Figures 4A and 4B). Consecutive measurements within the same segments taken at 4, 5, and 6 hours after FK506 administration showed no significant differences compared with preinjury (Figures 4A and 4B). Vascular reactivity to ACh at 10−7 mol/L and at 10−5 mol/L after FK506 administration also maintained increases of 10% and 20% in vessel diameter, respectively.

Figure 4.

Figure 4

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The vascular response to the chosen ACh concentrations reveals a significant difference between the employed monotherapies versus the combinational approach. The vascular reactivity to ACh at (A) 10−7 and (B) 10−5 mol/L in group 1 reveals the ∼10% and 20% increase in diameter, respectively. In groups 3 and 4, the vascular reactivity to ACh at each measurement time point after injury is significantly lower in both the contralateral and the ipsilateral sides compared with the values observed in group 1. However, in contrast to the vascular reactivity following either hypothermic intervention or FK506 administration, the vascular reactivity seen with the combination treatment in group 5 was well preserved, with no significant differences compared with group 1 at each time point. Values are expressed as mean±s.e.m. aSignificant differences compared with the values in all other groups at same time point (P<0.001). bSignificant differences compared with the values in groups 1 and 5 at same time point (P<0.001). cSignificant differences compared with corresponding value in the contralateral side (P<0.001). FK506, tacrolimus; LFPI, lateral fluid percussion injury.

Figure 5.

Figure 5

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The use of a combinational approach versus monotherapy results in a significant reduction of the burden of axonal damage in multiple brain loci. Bar graph shows a comparison of the mean density of APP-immunoreactive damaged axons in the corpus callosum, deep cortical layers of the mediodorsal neocortex, and dorsolateral thalamus among groups. Note that FK506 itself (group 1) exerted no significant axonal damage in comparison with the other groups. Also note that that when hypothermia was combined with FK506 (group 5), the numbers of damaged axons are greatly decreased in relation to the reduction of damaged axon in either hypothermic intervention (group 3) or FK506 administration (group 4). Values represent means±s.e.m. Statistical differences were analyzed by the Kruskal–Wallis test, followed by the Bonferroni test for multiple comparisons (*P<0.001, **P<0.005).

In group 2, which included animals subjected to LFPI with no treatment, vascular diameter could not be routinely measured because of the severe brain swelling that occurred on opening the cranial dura. However, one animal in this group could be evaluated, and in this case, no response to ACh at two concentrations was observed on either the contralateral or the ipsilateral side. Vascular reactivity to ACh at 10−7 mol/L at 4, 5, and 6 postinjury was −1.4%±1.1%, −2.1%±1.0%, and 0.6%±1.1%, respectively, on the contralateral side and −0.5%±0.5%, 1.2%±2.5%, and −0.5%±2.6%, respectively, on the ipsilateral side. Vascular reactivity to ACh at 10−5 mol/L was −1.2%±1.5%, −0.2%±1.5%, and −0.8%±0.5%, respectively, on the contralateral side and −0.7%±0.7%, −0.1%±3.4%, and −0.6%±0.6%, respectively, on the ipsilateral side.

Brain Arteriolar Reactivity after Lateral Fluid Percussion Injury with Treatment

In group 3, in which the resting diameters were 38±2.3 μm, 37±2.1 μm, and 37±2.2 μm at 4 to 6 hours postinjury on the contralateral side and 40±2.2 μm, 40±2.2 μm, and 40±2.3 μm at 4 to 6 hours postinjury on the ipsilateral side, the bilateral brain arteriolar reactivity to ACh at 10−7 mol/L and at 10−5 mol/L at 4 to 6 hours postinjury was significantly reduced compared with group 1 (group 3: ACh at 10−7 mol/L, 3.3%±1.0%, 3.1%±0.9%, and 2.4%±0.7% at 4 to 6 hours postinjury on the contralateral side; 1.2%±0.7%, 1.6%±0.7%, and 1.7%±0.6% at 4 to 6 hours postinjury on the ipsilateral side; ACh at 10−5 mol/L, 6.3%±1.5%, 8.1%±1.6%, and 6.2%±0.9% on the contralateral side; 3.0%±1.1%, 4.1%±1.4%, and 4.0%±1.1% on the ipsilateral side). In group 4, in which the contralateral resting diameters were 43±2.8 μm, 44±2.8 μm, and 45±2.8 μm at 4 to 6 hours postinjury, and the ipsilateral resting diameters were 39±1.8 μm, 40±1.7 μm, and 38±1.6 μm at 4 to 6 hours postinjury, the bilateral arteriolar reactivity to 10−7 and 10−5 mol/L ACh at 4 to 6 hours postinjury was again significantly reduced in comparison with group 1. In particular, in group 4, ACh at 10−7 mol/L triggered a dilation of 0.2%±0.2%, −1.2%±0.7%, and 0.3%±0.6% on the contralateral side and 0.8%±0.6%, −1.2%±0.6%, and 0.7%±0.6% on the ipsilateral side, whereas ACh at 10−5 mol/L resulted in a 1.0%±0.8%, −0.3%±0.7%, and 0.5%±0.9% dilation on the contralateral side and a 2.1%±0.6%, −0.9%±0.6%, and 0.5%±0.5% dilation on the ipsilateral side (Figures 4A and 4B). Although reduced, the overall vascular responses to ACh at two chosen concentrations after LFPI followed by hypothermia (group 3) were partially preserved on the contralateral side, with no statistically significant protection on the ipsilateral side (Figures 4A and 4B). In the case of FK506 (group 4), the reduced vascular reactivity approached extinction. In contrast, in group 5 (hypothermia plus FK506), vascular reactivity was preserved and significantly higher than that in groups 3 and 4. In group 5, in which the contralateral resting diameters were 40±2.7 μm, 41±2.5 μm, 40±2.4 μm at 4 to 6 hours postinjury, and the ipsilateral resting diameters were 40±2.5 μm, 39±2.5 μm, 40±2.3 μm at 4 to 6 hours postinjury, ACh at 10−7 mol/L elicited a 9.0%±0.6%, 9.8%±0.5%, and 10.2%±0.5% dilation at 4 to 6 hours postinjury on the contralateral side and a 7.3%±0.9%, 6.6%±0.8%, and 7.3%±0.6% dilation at 4 to 6 hours postinjury on the ipsilateral side with ACh at 10−5 mol/L eliciting a 20.7%±0.7%, 20.0%±1.0%, and 20.3%±0.8% dilation on the contralateral side and a 15.9%±1.4%, 15.9%±1.6%, and 15.4%±1.0% dilation on the ipsilateral side (Figures 4A and 4B). These values also illustrate that in group 5, vascular reactivity on the ipsilateral side was significantly lower at 5 and 6 hours postinjury using ACh 10−7 mol/L and at 4 and 6 hours postinjury using ACh 10−5 mol/L compared with the corresponding values on the contralateral side (Figures 4A and 4B).

Immunocytochemical Findings

Axonal Injury

Through quantitative analyses, the numbers of damaged callosal axons in the two areas sampled were sparse in group 1 (27±3 axons/mm2 and 44±5 axons/mm2, respectively) than in the other groups. These values reflecting background labeling were consistent with historical sham values (Figures 5A and 5B). In group 2 after LFPI, the numbers of callosal axons at 1.5 and 2.5 mm from the midline were striking and equaled 585±27 axons/mm2 and 809±47 axons/mm2, respectively (Figures 5A and 5B). This dramatic burden of axonal damage was in contrast to the significantly decreased axonal numbers found in group 5 (232±16 axons/mm2 and 562±46 axons/mm2, respectively), using the combination of hypothermia and FK506 (Figures 5A and 5B). When the numbers of damaged axons at 1.5 and 2.5 mm from the midline in group 2 were expressed at 100%, the numbers of damaged axons in these sectors in group 5 showed ∼60% and 30% reduction, respectively. This observation also contrasted with the numbers observed in group 3 (425±29 axons/mm2 and 710±32 axons/mm2, respectively) and in group 4 (366±25 axons/mm2 and 723±42 axons/mm2, respectively), in which either hypothermia or FK506 alone was used (Figures 5A and 5B).

In addition to these changes in the corpus callosum, similar changes were observed in the deep cortical layers and dorsolateral thalamus (Figures 5C and 5D). These loci were chosen because they allowed the assessment of axonal damage in the cortical and diencephalic gray before the axon's passage into the subcortical white matter and the thalamic peduncles. The numbers of damaged axons in group 1 (30±5 axons/mm2 and 12±2 axons/mm2 in the cortex and thalamus, respectively) were significantly lower than those in any other experimental group, reflecting background immunoreactivity. The numbers of damaged axons in group 5 (103±10 axons/mm2 and 65±7 axons/mm2) were significantly lower than those in group 2 (189±19 axons/mm2 and 154±10 axons/mm2), with a reduction of ∼45% and 60%, respectively, in the deep cortex and thalamus. The numbers of damaged axons in group 5 were also lower than those observed in group 3 (139±18 axons/mm2 and 115±12 axons/mm2) and in group 4 (158±13 axons/mm2 and 133±13 axons/mm2).

Blood–Brain Barrier Disruption

The area of IgG immunostaining in group 1 (3.3±0.4 mm2) was significantly lower than that found in any other experimental group (Figure 6). There were no significant differences in the area of IgG immunostaining between groups 2, 3, and 4 (10.5±0.7 mm2, 12.5±1.0 mm2, and 12.2±0.5 mm2, respectively). However, the area of IgG immunostaining in group 5 (19.4±1.2 mm2) was significantly higher than that in groups 2, 3, and 4 (Figure 6). Neither hypothermia nor FK506 administration prevented endogenous IgG extravasation. In addition, the combination of hypothermia and FK506 exacerbated BBB disruption (Figures 2H, 2I, and 6).

Figure 6.

Figure 6

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Bar graph shows a comparison of the mean area of IgG immunostaining among groups. It must be noted that neither hypothermic intervention nor FK506 administration prevented the spatial extravasation of endogenous IgG. In addition, even the combination therapy of hypothermic intervention and FK506 administration exerted no protection for blood–brain barrier disruption. Values represent means±s.e.m (*P<0.001). IgG, immunoglobulin G.

Discussion

This study shows that the coupling of delayed hypothermia with FK506 provides significant protection from TBI-induced diffuse axonal injury and impaired vascular reactivity, both of which are important features of human TBI. Importantly, these protective effects significantly exceed the limited benefits provided by the use of either of these agents alone.

Our observation that the LFPI significantly reduced vascular reactivity to varying concentrations of ACh is consistent with previous studies (Suehiro et al, 2003; Ueda et al, 2003; Wei et al, 2009). Similarly, the observation that delayed posttraumatic hypothermia provided limited vascular protection in terms of reactivity to ACh is also consistent with previous observations (Suehiro et al, 2003; Ueda et al, 2003; Wei et al, 2009). Of interest, however, was the finding primarily in group 5 that vascular reactivity and its responses to hypothermia and FK506 varied on the ipsilateral versus contralateral sides. Given that the side ipsilateral to the injury approximated the injury pulse, it most likely triggered more vascular damage/dysfunction than seen on the contralateral side. However, such sided differences were not detected in our previous studies using impact acceleration models of TBI, which generated more uniform vascular responses (Baranova et al, 2008).

Although the partial protective effects of hypothermia have been well documented (Suehiro et al, 2003; Ueda et al, 2003; Wei et al, 2009) and have been reaffirmed in the current investigation, the potential that FK506 may yield some protection of the cerebral microcirculation has not been reported previously. Most have assumed that endothelial and smooth muscle components have no FK506-mediated responses. However, this impression has recently been challenged by the finding that FK506 suppresses adhesion molecule expression in vascular endothelial cells and nitric oxide synthase in vascular smooth muscle cells (Sasakawa et al, 2005; Thomale et al, 2007). Although our finding that the use of FK506 alone exerted no cerebrovascular protection following TBI, it is possible that our delayed (90 minutes) administration paradigm minimized any potential protection. Furthermore, new studies suggest that endothelial superoxide generation can inhibit calcineurin signaling (Namgaladze et al, 2005). As it is well known that TBI is associated with oxygen-radical production within the cerebral microcirculation (Wei et al, 1981), it is possible that this inhibited calcineurin activity, explaining the lack of FK506 efficacy.

Although the singular use of hypothermia provided limited protection in terms of cerebrovascular responsiveness to ACh, with limited benefits provided by FK506 alone, our finding of dramatic vascular protection with the combination of hypothermia and FK506 must be considered intriguing. In previous communications, we noted similar beneficial/additive vascular effects with the use of hypothermia and superoxide dismutase (Baranova et al, 2008). We posited that this enhanced beneficial response was related to a hypothermia-induced reduction of brain metabolism coupled with a possible hypothermic blunting of oxygen-radical production, allowing the superoxide dismutase to optimally exert its effects (Globus et al, 1995; Bayir et al, 2009). In the current communication, similar arguments can be advanced, although from a somewhat different perspective. Again, the use of hypothermia could reduce overall brain vascular metabolism while also reducing drug clearance (Tortorici et al, 2007). Such reduced metabolism could also explain a reduction in oxygen-radical production, which is linked to many of the vascular abnormalities described in the current communication. In addition, as it has been shown that oxygen radicals can affect endothelial-linked, calcineurin-mediated events (Namgaladze et al, 2005), it is possible that the inhibition of oxygen radicals through hypothermia also allowed FK506 to exert its maximal protective effects. The loss of a potential protective pathway could also relate to the fact that FK506 is known to inhibit nitric oxide production by suppressing the calcineurin-mediated dephosphorylation of nitrogen oxide synthase through the FK506/FK-binding protein 12 complex (Dawson et al, 1993). In this scenario, the protective effects of combinational therapy could reside in FK506/hypothermic potentiation to result in the overall suppression of oxygen radicals and nitric oxide which, by its interaction with oxygen radicals, can generate even more damaging species (Globus et al, 1995; Sasaki et al, 2004).

In addition to our demonstration that the coupling of delayed hypothermic intervention and FK506 significantly preserved vascular reactivity following TBI, we also showed that this strategy provided a marked reduction of damaged axons within the corpus callosum, neocortex, and thalamus, which are important loci of axonal injury in human TBI (Povlishock, 1992). As with the above-described cerebrovascular studies, the benefits of the individual use of hypothermia or FK506 were limited, with the only significant preservation seen with the use of our combinational approach. Our observation that the use of hypothermia alone provided partial protection in the corpus callosum, neocortex, and thalamus is consistent with previous observations in the corticospinal and medial lemniscal systems (Koizumi and Povlishock, 1998), in which we also showed an attenuation of intraaxonal calpain-mediated proteolysis (Büki et al, 1999). We posited that hypothermia resulted in metabolic suppression and in the inhibition of other cysteine protease-regulatory pathways.

Our finding that FK506 alone had only a limited effect on axonal protection was not consistent with our previous studies using this immunophilin ligand. Previously, using FK506, we observed axonal protection within the corticospinal and medial lemniscal systems (Singleton et al, 2001; Marmarou and Povlishock, 2006). Although the rationale for the current lack of protection is not clear, it most likely relates to FK506's therapeutic window. In previous studies, the drug was administered 30 minutes before injury (Singleton et al, 2001; Marmarou and Povlishock, 2006). In contrast, a 90-minute postinjury interval was currently used. As FK506, by its suppression of calcineurin, has been recognized to modulate some of the processes involved in the pathogenesis of axonal swelling and disconnection (Singleton et al, 2001; Marmarou and Povlishock, 2006), it is conceivable that delayed drug administration could not reverse those events, which were underway at the time of drug administration. However, the fact that dramatic axonal protection was achieved using the combination of hypothermia and FK506 is intriguing. In this study, it is likely that hypothermia, by its inhibition of metabolism, blunts the progression of those intraaxonal cascades leading to axonal swelling and disconnection, with the caveat that this continued metabolic suppression and delayed axotomy now allow FK506 to exert its maximal effect, reducing axonal damage.

As noted, BBB findings associated with the combined use of FK506 and hypothermia showed no benefit of this combinational approach. In fact, it seemed to exacerbate barrier damage. This finding was unanticipated because in ischemia, the use of a similar combinational therapy reduced infarct volume and decreased brain edema (Nito et al, 2004). Although these ischemic studies involved no direct BBB assessment, it would not be unreasonable to posit that the reduction in brain edema would be associated with some preservation of BBB status. In contrast to these findings in ischemia, the use of FK506 in the context of traumatically induced contusion exerted no benefit (Thomale et al, 2007; Scheff and Sullivan, 1999). This suggests that its action in the context of TBI may be different from that seen with ischemia. As traumatically induced contusions have long been associated with disrupted BBB status, the failure of FK506 to protect against contusional expansion seems to be consistent with the observations of the current study showing a lack of BBB protection. Our observation that the combination of hypothermia and FK506 actually exacerbated BBB disruption is more difficult to explain. Conceivably, the preservation of vasoreactivity achieved using this combinational approach resulted in improved cerebral perfusion, thereby enhancing the perfusion pressure found in the area of barrier disruption. Alternatively, the additional possibility exists that hypothermia decreased the diffusion/clearance of the extracellular IgG, thereby providing an erroneous interpretation of the degree of BBB disruption. Obviously, this issue requires further investigation.

Conclusions

In summary, the current studies illustrate in animal models that hypothermia and FK506 alone exert limited axonal or/and vascular protection following TBI. Importantly, however, this protection was significantly enhanced by the coupling of hypothermic intervention with the use of delayed immunophilin ligand administration. These findings are consistent with our previous observations made in the microcirculation, suggesting that hypothermia alone not only provides protection in the brain and its vasculature but importantly also extends the therapeutic window over which drugs effective only during early post-TBI can regain maximal efficacy. Although the use of hypothermia in the treatment of traumatically brain-injured patients remains controversial and in fact, has been negatively impacted by the recent stoppage of a major clinical trial (Dr Guy Clifton, personal communication), the current study raises several interesting implications. First and foremost, although it seems that hypothermia may partially protect, its true benefit may reside in its ability to extend the therapeutic window for the use of other pharmacological/therapeutic approaches.

Acknowledgments

The authors thank Susan Walker and Lynn Davis for their excellent technical assistance.

The authors declare no conflict of interest.

Footnotes

This study is supported by NIH grants HD 055813 and NS 057175.

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