- Email: [email protected]
Hypertonic saline attenuates tissue loss and astrocyte hypertrophy in a model of traumatic brain injury
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Hypertonic saline attenuates tissue loss and astrocyte hypertrophy in a model of traumatic brain injury Melanie B. Elliott a,⁎, Jack J. Jallo a,1 , Mary F. Barbe c,2 , Ronald F. Tuma b,3 a
Department of Neurosurgery, Thomas Jefferson University, 1025 Walnut Street, Philadelphia, PA 19107, USA Department of Physiology, Temple University School of Medicine, Philadelphia, PA, USA c Department of Physical Therapy, Temple University, Philadelphia, PA, USA b
A R T I C LE I N FO
AB S T R A C T
Article history:
Hypertonic saline is currently being used in the treatment of patients with post-traumatic
Accepted 26 September 2009
cerebral edema and elevated intracranial pressure resulting from TBI. A limited number of
Available online 3 October 2009
studies show the cellular effects of hypertonic saline and no studies, to our knowledge, have investigated the effects on astrocytes. The role of astrocyte responses after traumatic brain
Keywords:
injury remains unclear. There is evidence that reduced astrocyte proliferation is detrimental
Hypertonic saline
while increased hypertrophy and proliferation are signs of increased injury severity.
Traumatic brain injury
Therefore, this study focused on the hypothesis that hypertonic saline-induced
Cortical contusion injury
improvements in histological outcome are time dependent and may be associated with
Tissue loss
alterations in astrocyte hypertrophy after cortical contusion injury. Histopathological
Astrocyte
changes at 7 days after controlled cortical impact (CCI) injury were examined. Brain tissue
Gliosis
loss determined using cresyl violet staining and astrocyte hypertrophy and proliferation were assessed using glial fibrillary acidic protein immunostaining in hypertonic saline and normal saline treated rats, and untreated, injured controls. Effects of the timing of hypertonic saline treatment administration on tissue loss were also examined. Plasma osmolarity and sodium levels were measured over 4 h and again at 24 h following hypertonic saline administration. Results show that hypertonic saline treatment reduced tissue loss that correlated with attenuated astrocyte hypertrophy characterized by reductions in astrocyte immunoreactivity without changes in the number of astrocytes after CCI injury. Delayed treatment of hypertonic saline resulted in the greatest reduction in tissue loss compared to all other treatments indicating that there is a therapeutic window for hypertonic saline use after traumatic brain injury. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Traumatic brain injury (TBI) results in an estimated 1.4 million deaths, hospitalizations, and emergency department
visits annually in the United States at an estimated annual cost of $60 billion (Langlois et al., 2006). Currently, approximately 5.3 million Americans are living with
⁎ Corresponding author. Fax: +1 215 503 1978. E-mail addresses: [email protected], [email protected] (M.B. Elliott), [email protected] (J.J. Jallo), [email protected] (M.F. Barbe), [email protected] (R.F. Tuma). 1 Thomas Jefferson University, 909 Walnut Street, 2nd Floor, Philadelphia, PA 19107, USA. Fax: +1 215 503 9170. 2 Temple University College of Health Professions, 3307 North Broad St., Philadelphia, PA 19140, USA. Fax: +1 215 707 7500. 3 Temple University School of Medicine, 3400 North Broad Street Old Medical School, 2nd Floor, Philadelphia, PA 19140, USA. Fax: +1 215 707 4003. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.09.104
184
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 –19 1
disabilities related to TBI. Adding to these costs is the evidence that TBI can cause epilepsy and increase the risk for conditions such as Alzheimer's disease, Parkinson's disease, and other brain disorders that become more prevalent with age (NINDS 2002). Osmotherapy agents such as hypertonic saline are currently being used in the treatment of patients with post-traumatic cerebral edema and elevated intracranial pressure resulting from TBI. Numerous human and animal studies have shown that administration of hypertonic saline decreases elevated intracranial pressure and cerebral edema following head injury, and improves cerebral hemodynamics (Shackford, 1997; Shackford et al., 1998; Simma et al., 1998; Horn et al., 1999; Khanna et al., 2000; Munar et al., 2000; Qureshi et al., 2002; Vialet et al., 2003; Ziai et al., 2007). Further evidence suggests hypertonic saline solutions are not only safe but in certain cases may be more beneficial than conventional fluid therapy such as mannitol (Anderson et al., 1997; Harutjunyan et al., 2005; Ware et al., 2005; Soustiel et al., 2006). While there is adequate evidence that hypertonic saline improves cerebral hemodynamics after injury (Ziai et al., 2007), studies that document the cellular effects of hypertonic saline are limited (Doyle et al., 2001; Thomale et al., 2004; Elliott et al., 2007; Sell et al., 2008). Hypertonic saline has been shown to have a favorable effect on histological and biochemical outcomes in a growing number of animal studies of CNS injury (Tuma et al., 1997; Sumas et al., 2001; Soustiel et al., 2006; Yilmaz et al., 2007; Sell et al., 2008). To the best of our knowledge, no studies have investigated the effects of hypertonic saline treatment on reactive astrocytes after TBI. The role of astrocyte responses after traumatic brain injury remains unclear. While completely eliminating astrocyte proliferation has been shown to be detrimental, increased astrocyte hypertrophy and proliferation, collectively referred to as astrogliosis, were dependent on increased injury severity (Myer et al., 2006; Saatman et al., 2008). Evidence supports a protective role of reactive astrocytes following brain injury, whereas the complexity of astrocyte responses to injury are acknowledged (Myer et al., 2006; Otani et al., 2006; White and Jakeman, 2008). Several proposed protective functions of reactive astrocytes include reduced excitotoxic death of neurons via a sodium-dependent glutamate transport mechanism, decreased cerebral edema by altered water transport, and attenuation of oxidative damage. The idea that glial scar formation impedes axonal regeneration remains controversial since reparative functions of reactive astrocytes have been shown (White and Jakeman, 2008). On the other hand, inflammatory and cytotoxic substances released by astrocytes following injury have been implicated as being potentially deleterious to neurons (Myer et al., 2006). Therefore, we investigated whether therapeutic modulation of tissue loss using hypertonic saline may be associated with changes in hypertrophic astrocyte responses to traumatic brain injury. Previously, our laboratory found hypertonic saline administration after controlled cortical impact (CCI) injury did not improve acute ischemic tissue damage evaluated by triphenyltetrazolium chloride (TTC) staining and even showed slightly adverse effects (Elliott et al., 2007). Evidence suggests that a longer survival time may be important to show a treatment effect especially for tissue loss (Tuma et al., 1997;
Sumas et al., 2001; Levene et al., 2007; Yilmaz et al., 2007; Sell et al., 2008). Therefore, the objective of this study was to determine whether hypertonic saline improved histological outcome at 7 days after TBI and whether tissue loss was associated with altered astrocyte reactivity defined by cell hypertrophy and proliferation. Using CCI injury in rats we examined the effects of hypertonic saline treatment on tissue loss assessed by cresyl violet staining and astrocyte reactivity assessed by percent area fraction of GFAP immunoreactivity, GFAP labeled cell counts and morphological changes. An experiment was conducted to determine whether the timing of hypertonic saline treatment administration was important for reducing tissue loss. Changes in plasma osmolarity and sodium levels were monitored after a single bolus of hypertonic saline over a 4-h time period and again at 24 h following hypertonic saline administration.
2.
Results
Tissue loss determined using cresyl violet at 7 days after CCI injury was significantly reduced in rats treated with hypertonic saline immediately after injury compared to untreated CCI injured control rats, p = 0.0075 and F = 5.65 (Fig. 1) The percent area fraction of GFAP immunoreactivity was significantly reduced with hypertonic saline treatment of CCI injured rats compared to normal saline treated rats and untreated CCI injured controls, ANOVA p < 0.0001 and F = 28.20 (Figs. 2 and 3). The number of GFAP+ cells (Fig. 2B) was significantly greater for CCI injured (p < 0.001), NS treated (p < 0.01), and HS treated (p < 0.05) rats compared to uninjured controls; however, there were no treatment effects among CCI injured rats. Morphological examination also revealed that reduced GFAP immunoreactivity appears to be a reduction in hypertrophied soma and processes of reactive astrocytes (Fig. 3D). The injured hemispheres for all groups (Figs. 3A, C, and D) show substantial hypertrophy and proliferation of GFAP+ positive astrocytes in comparison to the uninjured, contralateral hemisphere (Fig. 3B). The percentage of tissue
Fig. 1 – Tissue loss in cresyl violet stained tissues at 7 days after controlled cortical impact (CCI) injury was significantly reduced in rats treated with hypertonic saline (HS; n = 15) immediately after injury compared to untreated CCI injured controls (n = 7), **p < 0.01. Normal saline rats (n = 12) were not statistically different from the other groups.
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
185
Fig. 2 – All CCI injured groups showed significantly greater % area fraction of GFAP+ immunoreactive cells (A) compared to uninjured untreated normal controls (n = 5; a = p < 0.001). GFAP+ astrocyte immunoreactivity was significantly reduced with hypertonic saline (HS) treatment of CCI injured rats (n = 8) compared to untreated CCI injured controls (n = 5; b = p < 0.05) and normal saline treated rats (n = 6; c = p < 0.01). The number of GFAP+ cells (B) was significantly greater for CCI injured (a = p < 0.001), NS treated (b = p < 0.01), and HS treated (c = p < 0.05) rats compared to uninjured controls. loss was moderately correlated with the level of GFAP immunoreactivity (r = 0.53, p = 0.02, R squared = 0.28) (Fig. 4). Delayed hypertonic saline treatment at 1 h resulted in a
significantly smaller percentage of tissue loss compared to injured controls, whereas tissue loss after delayed normal saline treatment was not different than injured controls
Fig. 3 – Micrograph showing GFAP positive astrocytes following CCI injury in an untreated control rat's ipsilateral hemisphere (A) and contralateral hemisphere (B), a normal saline treated rat (C), and a hypertonic saline treated rat (D). Note the reduction in hypertrophied astrocytes in the hypertonic saline treated rat (D) compared to the untreated injured control (A) and normal saline treated rat (C). Inset of (B) shows a low power image of the injury site (*) and contralateral hemisphere under a 10× objective. White bar in (A) = 100 μm; (A) to (D) are under a 40× objective.
186
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 –19 1
Fig. 4 – The percentage of tissue loss in cresyl violet stained tissue was moderately correlated with the percent of reactive GFAP positive astrocytes (r = 0.53, p = 0.02).
(ANOVA p = 0.0077 and F = 6.580) (Fig. 5). Delayed hypertonic saline treatment resulted in the greatest reduction in tissue loss; however, there was no statistical difference between immediate and delayed hypertonic saline groups at 7 days after injury. Plasma osmolarity and sodium levels were significantly elevated from baseline at 10 min and 2, 3, and 4 h after a 4 ml/kg bolus of hypertonic saline followed by a return to baseline at 24 h (ANOVA p < 0.0001; F = 21.77 and F = 13.54, respectively) (Fig. 6). Mean plasma osmolarity at baseline was 292.4 ± 6.9 mOsm and at 10 min and 2, 3, 4 and 24 h following a bolus of 7.5% HS mean plasma osmolarity was 321.0 ± 2.7, 310.0 ± 5.7, 311.6 ± 6.5, 309.2 ± 4.0, and 294.8 ± 1.9 mOsm, respectively. Mean plasma sodium at baseline was 142.0 ± 3.4 and at 10 min and 2, 3, 4 and 24 h following a 4 ml/kg bolus of 7.5% HS mean plasma sodium was 153.2 ± 1.2, 148.5 ± 1.9, 148.6 ± 2.8, 147.4 ± 2.4, and 144.1 ± 1.4 mOsm, respectively. Although CCI injury produced a significant motor deficit, there were no statistically significant group differences between any experimental groups at 7 days (data not shown).
3.
ments with delayed treatments of hypertonic saline administered from 30 min to 1 h after injury (Soustiel et al., 2006; Yilmaz et al., 2007; Sell et al., 2008). A time dependent influence on mechanisms related to the development of secondary injury may be involved. In contradiction to our previous report, immediate administration of hypertonic saline did not worsen outcome in this study. These findings may be explained by the use of 2,3,5-triphenyltetrazolium chloride (TTC) staining in the previous study, which does not distinguish hibernating tissue that may later recover. TTC was previously used to assess injury volume by identifying enzymatically inactive brain tissue often caused by ischemia (Okuno et al., 2001; Elliott et al., 2007; Thomale et al., 2004). Cresyl violet staining used in the present study delineates the volume of necrotic tissue loss rather than the spread of ischemia-induced enzymatic changes. Differences in injury volumes between our past and present studies may also be explained by differences in the effects on early injury processes versus the effects on later progression and expansion of the injured area. CCI injury creates a focal lesion with a necrotic core in which some tissue loss occurs in the acute period (24 h) after injury that eventually progresses to a larger cavity by 7 days (Chen et al., 2003; Saatman et al., 2008). Injury volumes detected by TTC shrinks from 1 to 7 days indicating recovery from acute ischemic damage without treatment (Baskaya et al., 2000; Elliott et al., 2008b). TTC may overestimate infarct volume by including vital brain tissue that is enzymatically inactive (Okuno et al., 2001); however, TTC remains useful for evaluating agents that influence tissue perfusion. Combined, past and present findings indicate that even though there was a slight increase in ischemic changes acutely following hypertonic saline treatment compared to control, it does not translate into increased infarcted tissue. Our current results show that hypertonic saline treatment actually reduced tissue loss. Our results showed that a single bolus of 7.5% hypertonic saline maintained plasma osmolarity and sodium levels within clinical limits (320–360 mOsm/kg and 155–170 mM, respectively) (Knapp, 2005; Wenham et al., 2008). Plasma
Discussion
Findings from this study showed that hypertonic saline treatment reduced necrotic tissue loss in cresyl violet stained tissue 7 days after CCI injury. While the reduced tissue loss after hypertonic saline treatment was associated with reduced percent area fraction of GFAP immunoreactivity of hypertrophied astrocytes, there was no difference in the number of astrocytes with treatment. As reported in a previous investigation by our laboratory, the timing of hypertonic saline administration is important (Elliott et al., 2007). Delayed administration of hypertonic saline was the most effective treatment that resulted in the greatest reduction in tissue loss after CCI injury. In previous studies by our laboratory, the effects of hypertonic saline on outcome from traumatic brain injury and spinal cord injury were also dependent on the timing of treatment administration (Elliott et al., 2007; Tuma et al., 1997; Spera et al., 2000; Levene et al., 2007). Several other laboratories using models of head injury showed improve-
Fig. 5 – Delayed hypertonic saline treated rats (delayed HS; n = 4) resulted in a significantly smaller percentage of tissue loss in cresyl violet stained tissue compared to CCI injured controls at 7 days after injury (n = 6; *p < 0.01). Delayed normal saline rats (delayed NS; n = 3) were not statistically different from the other groups.
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
187
Fig. 6 – Serum osmolarity and sodium levels in rats (n = 5) given a single 4 ml/kg bolus of hypertonic saline at pre-treatment time 0 and post-treatment times 10 min, 2 h, 3 h, 4 h, and 24 h. Increased serum levels of each lasted 4 h (*p < 0.05 and **p < 0.01) compared to pre-treatment time 0 and were back to pre-treatment levels by 24 h.
osmolarity and sodium levels were significantly increased within 10 min of infusion and remained elevated above baseline values for at least 4 h. Anderson et al. (1997) showed that a 7.5% hypertonic saline bolus infusion significantly increased plasma osmolarity for 12 h and remained above baseline for 24 h. It is important to note that a single bolus injection of hypertonic saline has a prolonged effect on plasma osmolarity and sodium. In comparison to the osmotic effects of hypertonic saline, the osmotic effects of mannitol do not last. The critical flaws with mannitol use are rebound increases in ICP and blood–brain barrier disruption whereby its repeated or prolonged use is contraindicated (Knapp, 2005). Mannitol is the most commonly used osmotic agent as firstline therapy for combating cerebral edema and lowering elevated ICP (Qureshi and Suarez, 2000; Knapp, 2005; Wenham et al., 2008). However, hypertonic saline is often administered in addition to mannitol especially for rebound ICP and increased ICP resistant to mannitol, or instead of mannitol by some units (Wenham et al., 2008). Evidence that repeated hypertonic saline administration may be more effective and safer than mannitol supports the widely accepted use of repeated hypertonic saline (Knapp, 2005; Wenham et al., 2008). Treatment guidelines for the most effective hypertonic saline concentration or osmotic load are presently inadequate (Qureshi and Suarez, 2000). Therefore, future studies investigating repeated hypertonic saline administrations and comparing different hypertonic saline concentrations from a mechanistic approach are required in order to move towards improvements in the clinical management of TBI. It has been proposed that the neuroprotective effects of hypertonic saline extend beyond its osmotic and rheologic properties (Doyle et al., 2001; Knapp, 2005). Hypertonic saline has shown potential neurochemical and immunomodulatory effects that may offer neuroprotection (Doyle et al., 2001; Soustiel et al., 2006; Yilmaz et al., 2007). For example, hypertonic saline resulted in a greater reduction in neuroinflammation and apoptosis, independent of the infused volume, compared to normal saline and mannitol treatment of focal brain injury (Soustiel et al., 2006). To date, it has
remains controversial as to which type of osmotherapy solution offers superior neuroprotection after TBI. In the present study CCI injury produced substantial increases in the levels of GFAP immunoreactivity and number of GFAP+ astrocytes in the adjacent injured cortex indicative of astrogliosis. In this study astrogliosis refers to “reactive” astrocytes in closest proximity to the injury site that are characterized by increased expression of cellular markers like GFAP, altered morphology such as hypertrophy, cell proliferation, and formation of glial scarring (Hampton et al., 2004; White and Jakeman, 2008). In a spinal cord injury model, astrocytes described as “activated” appear distal to the injury site in response to environmental stimuli but do not produce glial scarring (White and Jakeman, 2008). Quantification and morphological assessments of GFAP+ astrocytes in CCI injured hemispheres adjacent to the injury site revealed both substantial hypertrophy and proliferation of soma and processes compared to quiescent astrocytes in the uninjured hemispheres. Other laboratories using a mouse CCI injury model showed astrocyte hypertrophy and proliferation at 7 days after injury that are similar to our results (Myer et al., 2006; Saatman et al., 2008). Hypertonic saline treatment significantly attenuated astrocyte hypertrophy as evidenced by the combined results for reduced GFAP immunoreactivity and no difference in the number of astrocytes after CCI injury. Although reduced, it should be noted that astrogliosis was still present in the hypertonic saline group compared to the uninjured control and uninjured contralateral hemisphere of CCI injured rats. Reduced astrocyte immunoreactivity but not proliferation was correlated with reduced tissue loss. Evidence that astrocytic reactivity after CNS injury has many beneficial but also potentially deleterious aspects has lead to some controversy on this topic (Ghirnikar et al., 1998; Chen et al., 2003; Chen and Swanson, 2003; Saatman et al., 2008). This debate over the role of reactive astrocytes is fueled by contradictions in the literature. For example, a neuroprotective role was recently demonstrated in which completely eliminating astrocyte cell division after moderate CCI injury increased neuronal loss. Similarly, a study using GFAP knockout mice found increased
188
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 –19 1
neuronal degeneration following TBI and excitotoxic insults compared to wild-type mice (Otani et al., 2006). In contrast, studies using the CCI injury model describe earlier and more sustained astrogliosis responses, as well as increased levels of astrocyte hypertrophy and proliferation that were dependent on increased injury severity (Chen et al., 2003; Myer et al., 2006; Saatman et al., 2008). Therefore, modulation of certain reactive astrocyte processes such as hypertrophy may be reflective of changes in injury severity or cell survival (Ghirnikar et al., 1998; Myer et al., 2006; Saatman et al., 2008; White and Jakeman, 2008). Hypertonic saline may influence one or more of the postulated mechanisms of TBI induced neuronal degeneration including neuroinflammation, excitotoxicity and oxidative damage. It has also been proposed that hypertonic saline has immunomodulatory properties (Junger et al., 1998; Spera et al., 1998; Rotstein, 2000; Doyle et al., 2001; Pascual et al., 2002; Soustiel et al., 2006). Production of inflammatory mediators after injury stimulates astrocyte reactivity (Chen and Swanson, 2003; Hampton et al., 2004; White and Jakeman, 2008). On the other hand, astrocytes recruit inflammatory cells to the injured site and are a cellular source of several inflammatory cytokines and chemokines (Sama et al., 2008). Recent attention has been focused on the relationship between neuroinflammation and excitotoxicity (Chang et al., 2008). For example, several reports have shown that proinflammatory cytokines IL-1 beta and TNF alpha inhibit glutamate transporters found on astrocytes contributing to neurotoxicity (Boycott et al., 2008; Prow and Irani, 2008; Sama et al., 2008). Hypertonic saline may enhance sodium-dependent glutamate transport thereby reducing glutamate excitotoxicity (Phillis et al., 1999; Chen and Swanson, 2003; Ziai et al., 2007). Improved glutamate handling or reduced neuroinflammation are likely candidates for altering hypertrophied astrocytes and enhancing neuron survival. Moreover, hypertonic saline has been shown to reduce oxidative damage after closed head trauma (Sell et al., 2008). Astrocytes are known to scavenge free radicals in which they are especially vulnerable to destruction when exposed to reactive oxygen species (Chen and Swanson, 2003). Future studies will target these proposed mechanisms of neuronal degeneration. In conclusion, our results show that hypertonic saline treatment reduced tissue loss in which the greatest benefit occurred with delayed administration indicating that there is a therapeutic window for the treatment of traumatic brain injury. Reduced tissue loss following hypertonic saline treatment correlated with reduced astrocyte hypertrophy, whereas astrocyte cell number was not affected by any treatment following CCI injury. In addition to osmotic and rheological effects on cerebral hemodynamics and components of the neurovascular unit and immune system, another effect of hypertonic saline is modulation of astrocyte hypertrophy.
4.
Experimental procedures
Prior to initiating any research, the Temple University Institutional Animal Care and Use Committee (IACUC) reviewed and approved the research protocol, and approved the use of Sprague–Dawley rats. Rats were housed in the
Temple University School of Medicine, Central Animal Facility accredited by an American Association for the Accreditation of Laboratory Animal Care, and complies with NIH standards. All animals were allowed to wake up from anesthesia. Postoperative care consisted of keeping the animals in separate cages with unrestricted food and water. Animals were euthanized with a lethal dose of pentobarbitol prior to any histological analysis.
4.1.
Traumatic brain injury
TBI was produced in male Sprague–Dawley rats (n = 62) weighing 300–400 g using a well known model, controlled cortical impact (CCI) injury described previously (Elliott et al., 2007, 2008b). The animals were deeply sedated with 1 ml/kg ketamine/xylazine (1:1). Body and cranial temperature was monitored and maintained at 37 °C and betadine was applied to prevent infection. A 6-mm craniectomy was performed using a high-speed drill (Champ-Air Dental Drill, Benco Dental, Wilkes-Barre, PA) over the right sensorimotor cortex, midway between lambda and bregma sutures. A pneumatic piston impactor device (Biomedical Engineering Facility, Virginia Commonwealth University, Richmond, VA) with a 5mm-diameter, rounded tip was used to create a moderate injury (2.0 mm depth, 4.0 m/s velocity, and 130 ms duration of impact). Following injury, the bone flap was replaced and sealed with bone wax, and the skin was sutured closed with 3.0 black braided silk sutures.
4.2.
Experimental design
Fifty-two rats in CCI injured groups were randomly assigned to receive 0.9% normal saline (NS; n = 12), 7.5% hypertonic saline (HS; n = 15), delayed NS (n = 3), delayed HS (n = 4), or no treatment (CCI control; n = 18). Five rats were uninjured, untreated controls (controls) for brain histological analyses and five rats were used for serum analyses. Fluid treated rats were infused with 4 ml/kg of either 7.5% NaCl or 0.9% NaCl normal saline through the femoral vein (polyethylene 10 catheter) using a syringe infusion pump (model 200; KD Scientific, Holliston, MA) at a rate of 0.2 ml/min. Immediate treatment began 10 min after injury, whereas delayed treatments were administered at 1 h after injury. Percent tissue loss determined using cresyl violet staining and GFAP positive immunoreactivity were quantified in a blinded fashion at 7 days after CCI injury. Statistical comparisons were performed using a one-way analysis of variance (ANOVA). Post hoc between group comparisons were performed using the Tukey's multiple comparison Test. Pearson correlation was used to evaluate the relationship between histological assessments. Significance levels were set at p < 0.05 for all statistical analyses and results are reported as the mean and SEM. All data were analyzed using the Prism statistical program.
4.3.
Histological analyses
Rats were euthanized with a lethal dose of intraperitoneal sodium pentobarbital (1.5 ml), then perfused transcardially with 200 ml normal saline followed by 200 ml ice-cold 4% paraformaldehyde (Sigma) for fixation. Brains were stored in 4% paraformaldehyde overnight at 4 °C, then transferred to a 30% sucrose solution for 3 days for cryoprotection. Brains were then sectioned coronally (20 μm) on a freezing microtome with approximately 400 μm distance between slices. Serial sections in a 1:10 series were used for histological analysis, mounted
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
onto gelatin coated slides and dried overnight. Sections were stained with 0.2% cresyl violet (Sigma Chemical, St. Louis, MO) for general histological examination and measurement of tissue loss. Alternate sections were stained with a monoclonal antibody against glial fibrillary acidic protein (GFAP; clone GA5; Sigma Chemical Co., St. Louis, MO) and biotinylated donkey anti-mouse IgG secondary antibody (Jackson ImmunoResearch) to assess changes in astrocyte morphology indicative of reactivity (hypertrophy and/or proliferation) and quantify changes in the percent area fraction (defined below) and number of GFAP+ immunoreactive cells. GFAP was visualized using DAB (Sigma) and hematoxylin (Gill-1; Sigma) was used as a counterstain. Stained sections were dehydrated in graded alcohol, and defatted in xylene (Fisher Scientific) before being coverslipped with Permount (Fisher Scientific). Cresyl violet stained sections were viewed using light microscopy for gross morphology (Olympus BX5) and photographed using a Nikon Coolpix 950 digital camera at 2× magnification. Percent tissue loss in cresyl violet stained tissue was measured using NIH ImageJ software and reported as the percent volume of tissue loss. The volume of tissue loss was calculated by multiplying the area of the tissue loss delineated by the absence of cresyl violet staining for each section by the thickness of each section, and summing the products. Area of tissue loss was measured by subtracting the area of remaining tissue in the ipsilateral hemisphere from the area of tissue in the contralateral hemisphere. Percent volume of tissue loss was calculated as the volume of tissue loss divided by the volume of the contralateral hemisphere multiplied by 100. Brain tissues were analyzed quantitatively for GFAP+ cells using a Nikon fluorescence microscope interfaced with a quantification system (Bioquant System, Bioquant, TN) and an X,Y motorized stage. Quiescent GFAP+ astrocytes and reactive GFAP+ astrocytes (evidenced by hypertrophied soma and processes and cell proliferation) were captured using A Retiga EXI cooled camera (QImaging, Surrey, BC). GFAP immunoreactivity was systematically quantified in a region of interest that included the sensorimotor cortex immediately adjacent to the injury extending to the sagittal sulcus using an irregular specialty measurement tool available with the image analysis system (Bioquant, Nashville, TN). GFAP+ immunoreactivity was reported as a percent area fraction as further described by Al-Shatti et al. (2005). The mean area fraction of thresholded immunoreactive product in the selected region of interest was determined by dividing the video count area of pixels above background thresholds by the total number of pixels in the entire chosen image field as described previously (Elliott et al., 2008a). Video count area is the number of pixels in an image field that meet a user defined criterion multiplied by the area of a pixel at the selected magnification (20× objective and 350× magnification factor for this analysis). GFAP percent area fraction determines the quantity of GFAP staining (number of pixels above threshold) appearing in a given area whereby reactive astrocytes can be identified, however, does not distinguish between astrocyte hypertrophy and proliferation. Therefore, high-power morphological evaluation and cell counting were also performed to determine whether hypertrophy (defined as increased length and/or girth of GFAP positive soma and processes) or proliferation (defined as increased cell number) was present. An independent random sampling approach described by Mouton (2002) was used to count the number of GFAP+ cells (Mouton, 2002). The mean number of GFAP+ labeled cells was counted in the ipsilateral cortex adjacent to the injury and closest to the midline under 100× magnification in which three measurements were made
189
per section. Every 10th serial section through the injury was counted in order to determine the mean number of GFAP+ cells in a set volume of tissue (0.27 mm3) per 3 sections. To avoid bias in estimating the mean number of GFAP+ labeled cells, only cells in which the nucleus (unlabeled) was visible were measured. All measurements for cresyl violet and GFAP analyses were performed in a blinded manner.
4.4.
Motor behavior assessment
A balance beam test for motor coordination and balance was conducted before and 7 days after CCI injury in all treatment and control groups (Feeney et al., 1982; Goldstein and Davis, 1990; Garcia et al., 1995; Sell et al., 2008). The balance beam (2 × 4 × 122 cm wooden beam at a height of 72 cm) has a rectangular box (32.5 × 20.5 × 18.0 cm) on one end that served as a motivational tool for a place of rest and comfort. Rats learned how to cross the balance beam prior to any scoring of trials usually within three trials. The scoring for the balance beam test ranges from 1 to 7 in which 1 represents the greatest motor deficit and 7 represents normal.
4.5.
Serum analysis
Sprague–Dawley Rats (n = 5) were anesthetized with xylazine and ketamine (1:1) and followed by cannulation of both the right and left femoral veins for fluid infusion and blood withdraw, respectively. Blood samples were collected with a syringe flushed with heparin at baseline prior to hypertonic saline infusion and following infusion of a single 4 ml/kg bolus of 7.5% HS. Serum was collected at 10 min and 2, 3, 4, and 24 h after infusion completion. Plasma osmolarity and sodium concentrations were measured using a Nova Blood Gas Analyzer.
Acknowledgments Support for this research was provided by the PA Department of Health and AHA (0415405U).
REFERENCES
Al-Shatti, T., Barr, A.E., Safadi, F.F., Amin, M., Barbe, M.F., 2005. Increase in inflammatory cytokines in median nerves in a rat model of repetitive motion injury. J. Neuroimmunol. 167, 13–22. Anderson, J.T., Wisner, D.H., Sullivan, P.E., Matteucci, M., Freshman, S., Hildreth, J., Wagner Jr., F.C., 1997. Initial small-volume hypertonic resuscitation of shock and brain injury: short- and long-term effects. J. Trauma 42, 592–600 discussion 600-591. Baskaya, M.K., Dogan, A., Temiz, C., Dempsey, R.J., 2000. Application of 2,3,5-triphenyltetrazolium chloride staining to evaluate injury volume after controlled cortical impact brain injury: role of brain edema in evolution of injury volume. J. Neurotrauma 17, 93–99. Boycott, H.E., Wilkinson, J.A., Boyle, J.P., Pearson, H.A., Peers, C., 2008. Differential involvement of TNF alpha in hypoxic suppression of astrocyte glutamate transporters. Glia 56, 998–1004. Chang, Y.C., Kim, H.W., Rapoport, S.I., Rao, J.S., 2008. Chronic NMDA administration increases neuroinflammatory markers
190
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 –19 1
in rat frontal cortex: cross-talk between excitotoxicity and neuroinflammation. Neurochem. Res. 33, 2318–2323. Chen, Y., Swanson, R.A., 2003. Astrocytes and brain injury. J. Cereb. Blood. Flow. Metab. 23, 137–149. Chen, S., Pickard, J.D., Harris, N.G., 2003. Time course of cellular pathology after controlled cortical impact injury. Exp. Neurol. 182, 87–102. Doyle, J.A., Davis, D.P., Hoyt, D.B., 2001. The use of hypertonic saline in the treatment of traumatic brain injury. J. Trauma Inj. Infect. Crit. Care 50, 367–383. Elliott, M.B., Jallo, J.J., Gaughan, J.P., Tuma, R.F., 2007. Effects of crystalloid–colloid solutions on traumatic brain injury. J. Neurotrauma 24, 195–202. Elliott, M.B., Barr, A.E., Kietrys, D.M., Al-Shatti, T., Amin, M., Barbe, M.F., 2008a. Peripheral neuritis and increased spinal cord neurochemicals are induced in a model of repetitive motion injury with low force and repetition exposure. Brain. Res. 1218, 103–113. Elliott, M.B., Jallo, J.J., Tuma, R.F., 2008b. An investigation of cerebral edema and injury volume assessments for controlled cortical impact injury. J. Neurosci. Methods 168, 320–324. Feeney, D.M., Gonzalez, A., Law, W.A., 1982. Amphetamine, haloperidol, and experience interact to affect rate of recovery after motor cortex injury. Science 217, 855–857. Garcia, J.H., Wagner, S., Liu, K.F., Hu, X.J., 1995. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation. Stroke 26, 627–634 discussion 635. Ghirnikar, R.S., Lee, Y.L., Eng, L.F., 1998. Inflammation in traumatic brain injury: role of cytokines and chemokines. Neurochem. Res. 23, 329–340. Goldstein, L.B., Davis, J.N., 1990. Beam-walking in rats: studies towards developing an animal model of functional recovery after brain injury. J. Neurosci. Methods 31, 101–107. Hampton, D.W., Rhodes, K.E., Zhao, C., Franklin, R.J., Fawcett, J.W., 2004. The responses of oligodendrocyte precursor cells, astrocytes and microglia to a cortical stab injury, in the brain. Neuroscience 127, 813–820. Harutjunyan, L., Holz, C., Rieger, A., Menzel, M., Grond, S., Soukup, J., 2005. Efficiency of 7.2% hypertonic saline hydroxyethyl starch 200/0.5 versus mannitol 15% in the treatment of increased intracranial pressure in neurosurgical patients—a randomized clinical trial [ISRCTN62699180]. Crit. Care 9, R530–R540. Horn, P., Munch, E., Vajkoczy, P., Herrmann, P., Quintel, M., Schilling, L., Schmiedek, P., Schurer, L., 1999. Hypertonic saline solution for control of elevated intracranial pressure in patients with exhausted response to mannitol and barbiturates. Neurol. Res. 21, 758–764. Junger, W.G., Hoyt, D.B., Davis, R.E., Herdon-Remelius, C., Namiki, S., Junger, H., Loomis, W., Altman, A., 1998. Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38. J. Clin. Invest. 101, 2768–2779. Khanna, S., Davis, D., Peterson, B., Fisher, B., Tung, H., O'Quigley, J., Deutsch, R., 2000. Use of hypertonic saline in the treatment of severe refractory posttraumatic intracranial hypertension in pediatric traumatic brain injury. Crit. Care Med. 28, 1144–1151. Knapp, J.M., 2005. Hyperosmolar therapy in the treatment of severe head injury in children: mannitol and hypertonic saline. AACN Clin. Issues 16, 199–211. Langlois, J.A., Rutland-Brown, W., Wald, M.M., 2006. The epidemiology and impact of traumatic brain injury: a brief overview. J. Head Trauma Rehabil. 21, 375–378. Levene, H.B., Erb, C.J., Gaughan, J.P., Loftus, C.M., Tuma, R.F., Jallo, J.I., 2007. Hypertonic saline as a treatment for acute spinal cord injury: effects on somatic and autonomic outcomes as observed in a mouse model. Clin. Neurosurg. 54, 213–219.
Mouton, P.R. (Ed.), 2002. Principles and practices of unbiased stereology. The Johns Hopkins University Press, Baltimore. Munar, F., Ferrer, A.M., de Nadal, M., Poca, M.A., Pedraza, S., Sahuquillo, J., Garnacho, A., 2000. Cerebral hemodynamic effects of 7.2% hypertonic saline in patients with head injury and raised intracranial pressure. J. Neurotrauma 17, 41–51. Myer, D.J., Gurkoff, G.G., Lee, S.M., Hovda, D.A., Sofroniew, M.V., 2006. Essential protective roles of reactive astrocytes in traumatic brain injury. Brain 129, 2761–2772. Okuno, S., Nakase, H., Sakaki, T., 2001. Comparative study of 2,3, 5-triphenyltetrazolium chloride (TTC) and hematoxylin–eosin staining for quantification of early brain ischemic injury in cats. Neurol. Res. 23, 657–661. Otani, N., Nawashiro, H., Fukui, S., Ooigawa, H., Ohsumi, A., Toyooka, T., Shima, K., Gomi, H., Brenner, M., 2006. Enhanced hippocampal neurodegeneration after traumatic or kainate excitotoxicity in GFAP-null mice. J. Clin. Neurosci. 13, 934–938. Pascual, J.L., Ferri, L.E., Seely, A.J., Campisi, G., Chaudhury, P., Giannias, B., Evans, D.C., Razek, T., Michel, R.P., Christou, N.V., 2002. Hypertonic saline resuscitation of hemorrhagic shock diminishes neutrophil rolling and adherence to endothelium and reduces in vivo vascular leakage. Ann. Surg. 236, 634–642. Phillis, J.W., Song, D., O'Regan, M.H., 1999. Effects of hyperosmolarity and ion substitutions on amino acid efflux from the ischemic rat cerebral cortex. Brain Res. 828, 1–11. Prow, N.A., Irani, D.N., 2008. The inflammatory cytokine, interleukin-1 beta, mediates loss of astroglial glutamate transport and drives excitotoxic motor neuron injury in the spinal cord during acute viral encephalomyelitis. J. Neurochem. 105, 1276–1286. Qureshi, A.I., Suarez, J.I., 2000. Use of hypertonic saline solutions in treatment of cerebral edema and intracranial hypertension. Crit. Care Med. 28, 3301–3310. Qureshi, A.I., Suri, M.F., Ringer, A.J., Guterman, L.R., Hopkins, L.N., 2002. Regional intraparenchymal pressure differences in experimental intracerebral hemorrhage: effect of hypertonic saline. Crit. Care. Med. 30, 435–441. Rotstein, O.D., 2000. Novel strategies for immunomodulation after trauma: revisiting hypertonic saline as a resuscitation strategy for hemorrhagic shock. J. Trauma Inj. Infect. Crit. Care 49, 580–583. Saatman, K.E., Duhaime, A.C., Bullock, R., Maas, A.I., Valadka, A., Manley, G.T., 2008. Classification of traumatic brain injury for targeted therapies. J. Neurotrauma 25, 719–738. Sama, M.A., Mathis, D.M., Furman, J.L., Abdul, H.M., Artiushin, I.A., Kraner, S.D., Norris, C.M., 2008. Interleukin-1beta-dependent signaling between astrocytes and neurons depends critically on astrocytic calcineurin/NFAT activity. J. Biol. Chem. 283, 21953–21964. Sell, S.L., Avila, M.A., Yu, G., Vergara, L., Prough, D.S., Grady, J.J., DeWitt, D.S., 2008. Hypertonic resuscitation improves neuronal and behavioral outcomes after traumatic brain injury plus hemorrhage. Anesthesiology 108, 873–881. Shackford, S.R., 1997. Effect of small-volume resuscitation on intracranial pressure and related cerebral variables. J. Trauma Inj. Infect. Crit. Care 42, 48S–53S. Shackford, S.R., Bourguignon, P.R., Wald, S.L., Rogers, F.B., Osler, T.M., Clark, D.E., 1998. Hypertonic saline resuscitation of patients with head injury: a prospective, randomized clinical trial. J. Trauma Inj. Infect. Crit. Care 44, 50–58. Simma, B., Burger, R., Falk, M., Sacher, P., Fanconi, S., 1998. A prospective, randomized, and controlled study of fluid management in children with severe head injury: lactated Ringer's solution versus hypertonic saline. Crit. Care Med. 26, 1265–1270. Soustiel, J.F., Vlodavsky, E., Zaaroor, M., 2006. Relative effects of mannitol and hypertonic saline on calpain activity, apoptosis and polymorphonuclear infiltration in traumatic focal brain injury. Brain Res. 1101, 136–144.
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
Spera, P.A., Arfors, K.E., Vasthare, U.S., Tuma, R.F., Young, W.F., 1998. Effect of hypertonic saline on leukocyte activity after spinal cord injury. Spine 23, 2444–2448 discussion 2448-2449. Spera, P.A., Vasthare, U.S., Tuma, R.F., Young, W.F., 2000. The effects of hypertonic saline on spinal cord blood flow following compression injury. Acta Neurochir. (Wien) 142, 811–817. Sumas, M.E., Legos, J.J., Nathan, D., Lamperti, A.A., Tuma, R.F., Young, W.F., 2001. Tonicity of resuscitative fluids influences outcome after spinal cord injury. Neurosurgery 48, 167–172 discussion 172-163. Thomale, U., Griebenow, M., Kroppenstedt, S., Unterberg, A.W., Strover, J.F., 2004. Small volume resuscitation with hyperhaes improves pericontusional perfusion and reduces lesion volume following controlled cortical impact injury in rats. J. Neurotrauma 21, 1737–1746. Tuma, R.F., Vasthare, U.S., Arfors, K.E., Young, W.F., 1997. Hypertonic saline administration attenuates spinal cord injury. J. Trauma 42, S54–60. Vialet, R., Albanese, J., Thomachot, L., Antonini, F., Bourgouin, A., Alliez, B., Martin, C., 2003. Isovolume hypertonic solutes (sodium chloride or mannitol) in the treatment of refractory
191
posttraumatic intracranial hypertension: 2 mL/kg 7.5% saline is more effective than 2 mL/kg 20% mannitol. Crit. Care Med. 31, 1683–1687. Ware, M.L., Nemani, V.M., Meeker, M., Lee, C., Morabito, D.J., Manley, G.T., 2005. Effects of 23.4% sodium chloride solution in reducing intracranial pressure in patients with traumatic brain injury: a preliminary study. Neurosurgery 57, 727–736 discussion 727-736. Wenham, T.N., Hormis, A.P., Andrzejowski, J.C., 2008. Hypertonic saline after traumatic brain injury in UK neuro-critical care practice. Anaesthesia 63, 558–559. White, R.E., Jakeman, L.B., 2008. Don't fence me in: harnessing the beneficial roles of astrocytes for spinal cord repair. Restor. Neurol. Neurosci. 26, 197–214. Yilmaz, N., Dulger, H., Kiymaz, N., Yilmaz, C., Gudu, B.O., Demir, I., 2007. Activity of mannitol and hypertonic saline therapy on the oxidant and antioxidant system during the acute term after traumatic brain injury in the rats. Brain Res. 1164, 132–135. Ziai, W.C., Toung, T.J., Bhardwaj, A., 2007. Hypertonic saline: first-line therapy for cerebral edema? J. Neurol. Sci. 261, 157–166.
available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Hypertonic saline attenuates tissue loss and astrocyte hypertrophy in a model of traumatic brain injury Melanie B. Elliott a,⁎, Jack J. Jallo a,1 , Mary F. Barbe c,2 , Ronald F. Tuma b,3 a
Department of Neurosurgery, Thomas Jefferson University, 1025 Walnut Street, Philadelphia, PA 19107, USA Department of Physiology, Temple University School of Medicine, Philadelphia, PA, USA c Department of Physical Therapy, Temple University, Philadelphia, PA, USA b
A R T I C LE I N FO
AB S T R A C T
Article history:
Hypertonic saline is currently being used in the treatment of patients with post-traumatic
Accepted 26 September 2009
cerebral edema and elevated intracranial pressure resulting from TBI. A limited number of
Available online 3 October 2009
studies show the cellular effects of hypertonic saline and no studies, to our knowledge, have investigated the effects on astrocytes. The role of astrocyte responses after traumatic brain
Keywords:
injury remains unclear. There is evidence that reduced astrocyte proliferation is detrimental
Hypertonic saline
while increased hypertrophy and proliferation are signs of increased injury severity.
Traumatic brain injury
Therefore, this study focused on the hypothesis that hypertonic saline-induced
Cortical contusion injury
improvements in histological outcome are time dependent and may be associated with
Tissue loss
alterations in astrocyte hypertrophy after cortical contusion injury. Histopathological
Astrocyte
changes at 7 days after controlled cortical impact (CCI) injury were examined. Brain tissue
Gliosis
loss determined using cresyl violet staining and astrocyte hypertrophy and proliferation were assessed using glial fibrillary acidic protein immunostaining in hypertonic saline and normal saline treated rats, and untreated, injured controls. Effects of the timing of hypertonic saline treatment administration on tissue loss were also examined. Plasma osmolarity and sodium levels were measured over 4 h and again at 24 h following hypertonic saline administration. Results show that hypertonic saline treatment reduced tissue loss that correlated with attenuated astrocyte hypertrophy characterized by reductions in astrocyte immunoreactivity without changes in the number of astrocytes after CCI injury. Delayed treatment of hypertonic saline resulted in the greatest reduction in tissue loss compared to all other treatments indicating that there is a therapeutic window for hypertonic saline use after traumatic brain injury. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Traumatic brain injury (TBI) results in an estimated 1.4 million deaths, hospitalizations, and emergency department
visits annually in the United States at an estimated annual cost of $60 billion (Langlois et al., 2006). Currently, approximately 5.3 million Americans are living with
⁎ Corresponding author. Fax: +1 215 503 1978. E-mail addresses: [email protected], [email protected] (M.B. Elliott), [email protected] (J.J. Jallo), [email protected] (M.F. Barbe), [email protected] (R.F. Tuma). 1 Thomas Jefferson University, 909 Walnut Street, 2nd Floor, Philadelphia, PA 19107, USA. Fax: +1 215 503 9170. 2 Temple University College of Health Professions, 3307 North Broad St., Philadelphia, PA 19140, USA. Fax: +1 215 707 7500. 3 Temple University School of Medicine, 3400 North Broad Street Old Medical School, 2nd Floor, Philadelphia, PA 19140, USA. Fax: +1 215 707 4003. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.09.104
184
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 –19 1
disabilities related to TBI. Adding to these costs is the evidence that TBI can cause epilepsy and increase the risk for conditions such as Alzheimer's disease, Parkinson's disease, and other brain disorders that become more prevalent with age (NINDS 2002). Osmotherapy agents such as hypertonic saline are currently being used in the treatment of patients with post-traumatic cerebral edema and elevated intracranial pressure resulting from TBI. Numerous human and animal studies have shown that administration of hypertonic saline decreases elevated intracranial pressure and cerebral edema following head injury, and improves cerebral hemodynamics (Shackford, 1997; Shackford et al., 1998; Simma et al., 1998; Horn et al., 1999; Khanna et al., 2000; Munar et al., 2000; Qureshi et al., 2002; Vialet et al., 2003; Ziai et al., 2007). Further evidence suggests hypertonic saline solutions are not only safe but in certain cases may be more beneficial than conventional fluid therapy such as mannitol (Anderson et al., 1997; Harutjunyan et al., 2005; Ware et al., 2005; Soustiel et al., 2006). While there is adequate evidence that hypertonic saline improves cerebral hemodynamics after injury (Ziai et al., 2007), studies that document the cellular effects of hypertonic saline are limited (Doyle et al., 2001; Thomale et al., 2004; Elliott et al., 2007; Sell et al., 2008). Hypertonic saline has been shown to have a favorable effect on histological and biochemical outcomes in a growing number of animal studies of CNS injury (Tuma et al., 1997; Sumas et al., 2001; Soustiel et al., 2006; Yilmaz et al., 2007; Sell et al., 2008). To the best of our knowledge, no studies have investigated the effects of hypertonic saline treatment on reactive astrocytes after TBI. The role of astrocyte responses after traumatic brain injury remains unclear. While completely eliminating astrocyte proliferation has been shown to be detrimental, increased astrocyte hypertrophy and proliferation, collectively referred to as astrogliosis, were dependent on increased injury severity (Myer et al., 2006; Saatman et al., 2008). Evidence supports a protective role of reactive astrocytes following brain injury, whereas the complexity of astrocyte responses to injury are acknowledged (Myer et al., 2006; Otani et al., 2006; White and Jakeman, 2008). Several proposed protective functions of reactive astrocytes include reduced excitotoxic death of neurons via a sodium-dependent glutamate transport mechanism, decreased cerebral edema by altered water transport, and attenuation of oxidative damage. The idea that glial scar formation impedes axonal regeneration remains controversial since reparative functions of reactive astrocytes have been shown (White and Jakeman, 2008). On the other hand, inflammatory and cytotoxic substances released by astrocytes following injury have been implicated as being potentially deleterious to neurons (Myer et al., 2006). Therefore, we investigated whether therapeutic modulation of tissue loss using hypertonic saline may be associated with changes in hypertrophic astrocyte responses to traumatic brain injury. Previously, our laboratory found hypertonic saline administration after controlled cortical impact (CCI) injury did not improve acute ischemic tissue damage evaluated by triphenyltetrazolium chloride (TTC) staining and even showed slightly adverse effects (Elliott et al., 2007). Evidence suggests that a longer survival time may be important to show a treatment effect especially for tissue loss (Tuma et al., 1997;
Sumas et al., 2001; Levene et al., 2007; Yilmaz et al., 2007; Sell et al., 2008). Therefore, the objective of this study was to determine whether hypertonic saline improved histological outcome at 7 days after TBI and whether tissue loss was associated with altered astrocyte reactivity defined by cell hypertrophy and proliferation. Using CCI injury in rats we examined the effects of hypertonic saline treatment on tissue loss assessed by cresyl violet staining and astrocyte reactivity assessed by percent area fraction of GFAP immunoreactivity, GFAP labeled cell counts and morphological changes. An experiment was conducted to determine whether the timing of hypertonic saline treatment administration was important for reducing tissue loss. Changes in plasma osmolarity and sodium levels were monitored after a single bolus of hypertonic saline over a 4-h time period and again at 24 h following hypertonic saline administration.
2.
Results
Tissue loss determined using cresyl violet at 7 days after CCI injury was significantly reduced in rats treated with hypertonic saline immediately after injury compared to untreated CCI injured control rats, p = 0.0075 and F = 5.65 (Fig. 1) The percent area fraction of GFAP immunoreactivity was significantly reduced with hypertonic saline treatment of CCI injured rats compared to normal saline treated rats and untreated CCI injured controls, ANOVA p < 0.0001 and F = 28.20 (Figs. 2 and 3). The number of GFAP+ cells (Fig. 2B) was significantly greater for CCI injured (p < 0.001), NS treated (p < 0.01), and HS treated (p < 0.05) rats compared to uninjured controls; however, there were no treatment effects among CCI injured rats. Morphological examination also revealed that reduced GFAP immunoreactivity appears to be a reduction in hypertrophied soma and processes of reactive astrocytes (Fig. 3D). The injured hemispheres for all groups (Figs. 3A, C, and D) show substantial hypertrophy and proliferation of GFAP+ positive astrocytes in comparison to the uninjured, contralateral hemisphere (Fig. 3B). The percentage of tissue
Fig. 1 – Tissue loss in cresyl violet stained tissues at 7 days after controlled cortical impact (CCI) injury was significantly reduced in rats treated with hypertonic saline (HS; n = 15) immediately after injury compared to untreated CCI injured controls (n = 7), **p < 0.01. Normal saline rats (n = 12) were not statistically different from the other groups.
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
185
Fig. 2 – All CCI injured groups showed significantly greater % area fraction of GFAP+ immunoreactive cells (A) compared to uninjured untreated normal controls (n = 5; a = p < 0.001). GFAP+ astrocyte immunoreactivity was significantly reduced with hypertonic saline (HS) treatment of CCI injured rats (n = 8) compared to untreated CCI injured controls (n = 5; b = p < 0.05) and normal saline treated rats (n = 6; c = p < 0.01). The number of GFAP+ cells (B) was significantly greater for CCI injured (a = p < 0.001), NS treated (b = p < 0.01), and HS treated (c = p < 0.05) rats compared to uninjured controls. loss was moderately correlated with the level of GFAP immunoreactivity (r = 0.53, p = 0.02, R squared = 0.28) (Fig. 4). Delayed hypertonic saline treatment at 1 h resulted in a
significantly smaller percentage of tissue loss compared to injured controls, whereas tissue loss after delayed normal saline treatment was not different than injured controls
Fig. 3 – Micrograph showing GFAP positive astrocytes following CCI injury in an untreated control rat's ipsilateral hemisphere (A) and contralateral hemisphere (B), a normal saline treated rat (C), and a hypertonic saline treated rat (D). Note the reduction in hypertrophied astrocytes in the hypertonic saline treated rat (D) compared to the untreated injured control (A) and normal saline treated rat (C). Inset of (B) shows a low power image of the injury site (*) and contralateral hemisphere under a 10× objective. White bar in (A) = 100 μm; (A) to (D) are under a 40× objective.
186
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 –19 1
Fig. 4 – The percentage of tissue loss in cresyl violet stained tissue was moderately correlated with the percent of reactive GFAP positive astrocytes (r = 0.53, p = 0.02).
(ANOVA p = 0.0077 and F = 6.580) (Fig. 5). Delayed hypertonic saline treatment resulted in the greatest reduction in tissue loss; however, there was no statistical difference between immediate and delayed hypertonic saline groups at 7 days after injury. Plasma osmolarity and sodium levels were significantly elevated from baseline at 10 min and 2, 3, and 4 h after a 4 ml/kg bolus of hypertonic saline followed by a return to baseline at 24 h (ANOVA p < 0.0001; F = 21.77 and F = 13.54, respectively) (Fig. 6). Mean plasma osmolarity at baseline was 292.4 ± 6.9 mOsm and at 10 min and 2, 3, 4 and 24 h following a bolus of 7.5% HS mean plasma osmolarity was 321.0 ± 2.7, 310.0 ± 5.7, 311.6 ± 6.5, 309.2 ± 4.0, and 294.8 ± 1.9 mOsm, respectively. Mean plasma sodium at baseline was 142.0 ± 3.4 and at 10 min and 2, 3, 4 and 24 h following a 4 ml/kg bolus of 7.5% HS mean plasma sodium was 153.2 ± 1.2, 148.5 ± 1.9, 148.6 ± 2.8, 147.4 ± 2.4, and 144.1 ± 1.4 mOsm, respectively. Although CCI injury produced a significant motor deficit, there were no statistically significant group differences between any experimental groups at 7 days (data not shown).
3.
ments with delayed treatments of hypertonic saline administered from 30 min to 1 h after injury (Soustiel et al., 2006; Yilmaz et al., 2007; Sell et al., 2008). A time dependent influence on mechanisms related to the development of secondary injury may be involved. In contradiction to our previous report, immediate administration of hypertonic saline did not worsen outcome in this study. These findings may be explained by the use of 2,3,5-triphenyltetrazolium chloride (TTC) staining in the previous study, which does not distinguish hibernating tissue that may later recover. TTC was previously used to assess injury volume by identifying enzymatically inactive brain tissue often caused by ischemia (Okuno et al., 2001; Elliott et al., 2007; Thomale et al., 2004). Cresyl violet staining used in the present study delineates the volume of necrotic tissue loss rather than the spread of ischemia-induced enzymatic changes. Differences in injury volumes between our past and present studies may also be explained by differences in the effects on early injury processes versus the effects on later progression and expansion of the injured area. CCI injury creates a focal lesion with a necrotic core in which some tissue loss occurs in the acute period (24 h) after injury that eventually progresses to a larger cavity by 7 days (Chen et al., 2003; Saatman et al., 2008). Injury volumes detected by TTC shrinks from 1 to 7 days indicating recovery from acute ischemic damage without treatment (Baskaya et al., 2000; Elliott et al., 2008b). TTC may overestimate infarct volume by including vital brain tissue that is enzymatically inactive (Okuno et al., 2001); however, TTC remains useful for evaluating agents that influence tissue perfusion. Combined, past and present findings indicate that even though there was a slight increase in ischemic changes acutely following hypertonic saline treatment compared to control, it does not translate into increased infarcted tissue. Our current results show that hypertonic saline treatment actually reduced tissue loss. Our results showed that a single bolus of 7.5% hypertonic saline maintained plasma osmolarity and sodium levels within clinical limits (320–360 mOsm/kg and 155–170 mM, respectively) (Knapp, 2005; Wenham et al., 2008). Plasma
Discussion
Findings from this study showed that hypertonic saline treatment reduced necrotic tissue loss in cresyl violet stained tissue 7 days after CCI injury. While the reduced tissue loss after hypertonic saline treatment was associated with reduced percent area fraction of GFAP immunoreactivity of hypertrophied astrocytes, there was no difference in the number of astrocytes with treatment. As reported in a previous investigation by our laboratory, the timing of hypertonic saline administration is important (Elliott et al., 2007). Delayed administration of hypertonic saline was the most effective treatment that resulted in the greatest reduction in tissue loss after CCI injury. In previous studies by our laboratory, the effects of hypertonic saline on outcome from traumatic brain injury and spinal cord injury were also dependent on the timing of treatment administration (Elliott et al., 2007; Tuma et al., 1997; Spera et al., 2000; Levene et al., 2007). Several other laboratories using models of head injury showed improve-
Fig. 5 – Delayed hypertonic saline treated rats (delayed HS; n = 4) resulted in a significantly smaller percentage of tissue loss in cresyl violet stained tissue compared to CCI injured controls at 7 days after injury (n = 6; *p < 0.01). Delayed normal saline rats (delayed NS; n = 3) were not statistically different from the other groups.
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
187
Fig. 6 – Serum osmolarity and sodium levels in rats (n = 5) given a single 4 ml/kg bolus of hypertonic saline at pre-treatment time 0 and post-treatment times 10 min, 2 h, 3 h, 4 h, and 24 h. Increased serum levels of each lasted 4 h (*p < 0.05 and **p < 0.01) compared to pre-treatment time 0 and were back to pre-treatment levels by 24 h.
osmolarity and sodium levels were significantly increased within 10 min of infusion and remained elevated above baseline values for at least 4 h. Anderson et al. (1997) showed that a 7.5% hypertonic saline bolus infusion significantly increased plasma osmolarity for 12 h and remained above baseline for 24 h. It is important to note that a single bolus injection of hypertonic saline has a prolonged effect on plasma osmolarity and sodium. In comparison to the osmotic effects of hypertonic saline, the osmotic effects of mannitol do not last. The critical flaws with mannitol use are rebound increases in ICP and blood–brain barrier disruption whereby its repeated or prolonged use is contraindicated (Knapp, 2005). Mannitol is the most commonly used osmotic agent as firstline therapy for combating cerebral edema and lowering elevated ICP (Qureshi and Suarez, 2000; Knapp, 2005; Wenham et al., 2008). However, hypertonic saline is often administered in addition to mannitol especially for rebound ICP and increased ICP resistant to mannitol, or instead of mannitol by some units (Wenham et al., 2008). Evidence that repeated hypertonic saline administration may be more effective and safer than mannitol supports the widely accepted use of repeated hypertonic saline (Knapp, 2005; Wenham et al., 2008). Treatment guidelines for the most effective hypertonic saline concentration or osmotic load are presently inadequate (Qureshi and Suarez, 2000). Therefore, future studies investigating repeated hypertonic saline administrations and comparing different hypertonic saline concentrations from a mechanistic approach are required in order to move towards improvements in the clinical management of TBI. It has been proposed that the neuroprotective effects of hypertonic saline extend beyond its osmotic and rheologic properties (Doyle et al., 2001; Knapp, 2005). Hypertonic saline has shown potential neurochemical and immunomodulatory effects that may offer neuroprotection (Doyle et al., 2001; Soustiel et al., 2006; Yilmaz et al., 2007). For example, hypertonic saline resulted in a greater reduction in neuroinflammation and apoptosis, independent of the infused volume, compared to normal saline and mannitol treatment of focal brain injury (Soustiel et al., 2006). To date, it has
remains controversial as to which type of osmotherapy solution offers superior neuroprotection after TBI. In the present study CCI injury produced substantial increases in the levels of GFAP immunoreactivity and number of GFAP+ astrocytes in the adjacent injured cortex indicative of astrogliosis. In this study astrogliosis refers to “reactive” astrocytes in closest proximity to the injury site that are characterized by increased expression of cellular markers like GFAP, altered morphology such as hypertrophy, cell proliferation, and formation of glial scarring (Hampton et al., 2004; White and Jakeman, 2008). In a spinal cord injury model, astrocytes described as “activated” appear distal to the injury site in response to environmental stimuli but do not produce glial scarring (White and Jakeman, 2008). Quantification and morphological assessments of GFAP+ astrocytes in CCI injured hemispheres adjacent to the injury site revealed both substantial hypertrophy and proliferation of soma and processes compared to quiescent astrocytes in the uninjured hemispheres. Other laboratories using a mouse CCI injury model showed astrocyte hypertrophy and proliferation at 7 days after injury that are similar to our results (Myer et al., 2006; Saatman et al., 2008). Hypertonic saline treatment significantly attenuated astrocyte hypertrophy as evidenced by the combined results for reduced GFAP immunoreactivity and no difference in the number of astrocytes after CCI injury. Although reduced, it should be noted that astrogliosis was still present in the hypertonic saline group compared to the uninjured control and uninjured contralateral hemisphere of CCI injured rats. Reduced astrocyte immunoreactivity but not proliferation was correlated with reduced tissue loss. Evidence that astrocytic reactivity after CNS injury has many beneficial but also potentially deleterious aspects has lead to some controversy on this topic (Ghirnikar et al., 1998; Chen et al., 2003; Chen and Swanson, 2003; Saatman et al., 2008). This debate over the role of reactive astrocytes is fueled by contradictions in the literature. For example, a neuroprotective role was recently demonstrated in which completely eliminating astrocyte cell division after moderate CCI injury increased neuronal loss. Similarly, a study using GFAP knockout mice found increased
188
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 –19 1
neuronal degeneration following TBI and excitotoxic insults compared to wild-type mice (Otani et al., 2006). In contrast, studies using the CCI injury model describe earlier and more sustained astrogliosis responses, as well as increased levels of astrocyte hypertrophy and proliferation that were dependent on increased injury severity (Chen et al., 2003; Myer et al., 2006; Saatman et al., 2008). Therefore, modulation of certain reactive astrocyte processes such as hypertrophy may be reflective of changes in injury severity or cell survival (Ghirnikar et al., 1998; Myer et al., 2006; Saatman et al., 2008; White and Jakeman, 2008). Hypertonic saline may influence one or more of the postulated mechanisms of TBI induced neuronal degeneration including neuroinflammation, excitotoxicity and oxidative damage. It has also been proposed that hypertonic saline has immunomodulatory properties (Junger et al., 1998; Spera et al., 1998; Rotstein, 2000; Doyle et al., 2001; Pascual et al., 2002; Soustiel et al., 2006). Production of inflammatory mediators after injury stimulates astrocyte reactivity (Chen and Swanson, 2003; Hampton et al., 2004; White and Jakeman, 2008). On the other hand, astrocytes recruit inflammatory cells to the injured site and are a cellular source of several inflammatory cytokines and chemokines (Sama et al., 2008). Recent attention has been focused on the relationship between neuroinflammation and excitotoxicity (Chang et al., 2008). For example, several reports have shown that proinflammatory cytokines IL-1 beta and TNF alpha inhibit glutamate transporters found on astrocytes contributing to neurotoxicity (Boycott et al., 2008; Prow and Irani, 2008; Sama et al., 2008). Hypertonic saline may enhance sodium-dependent glutamate transport thereby reducing glutamate excitotoxicity (Phillis et al., 1999; Chen and Swanson, 2003; Ziai et al., 2007). Improved glutamate handling or reduced neuroinflammation are likely candidates for altering hypertrophied astrocytes and enhancing neuron survival. Moreover, hypertonic saline has been shown to reduce oxidative damage after closed head trauma (Sell et al., 2008). Astrocytes are known to scavenge free radicals in which they are especially vulnerable to destruction when exposed to reactive oxygen species (Chen and Swanson, 2003). Future studies will target these proposed mechanisms of neuronal degeneration. In conclusion, our results show that hypertonic saline treatment reduced tissue loss in which the greatest benefit occurred with delayed administration indicating that there is a therapeutic window for the treatment of traumatic brain injury. Reduced tissue loss following hypertonic saline treatment correlated with reduced astrocyte hypertrophy, whereas astrocyte cell number was not affected by any treatment following CCI injury. In addition to osmotic and rheological effects on cerebral hemodynamics and components of the neurovascular unit and immune system, another effect of hypertonic saline is modulation of astrocyte hypertrophy.
4.
Experimental procedures
Prior to initiating any research, the Temple University Institutional Animal Care and Use Committee (IACUC) reviewed and approved the research protocol, and approved the use of Sprague–Dawley rats. Rats were housed in the
Temple University School of Medicine, Central Animal Facility accredited by an American Association for the Accreditation of Laboratory Animal Care, and complies with NIH standards. All animals were allowed to wake up from anesthesia. Postoperative care consisted of keeping the animals in separate cages with unrestricted food and water. Animals were euthanized with a lethal dose of pentobarbitol prior to any histological analysis.
4.1.
Traumatic brain injury
TBI was produced in male Sprague–Dawley rats (n = 62) weighing 300–400 g using a well known model, controlled cortical impact (CCI) injury described previously (Elliott et al., 2007, 2008b). The animals were deeply sedated with 1 ml/kg ketamine/xylazine (1:1). Body and cranial temperature was monitored and maintained at 37 °C and betadine was applied to prevent infection. A 6-mm craniectomy was performed using a high-speed drill (Champ-Air Dental Drill, Benco Dental, Wilkes-Barre, PA) over the right sensorimotor cortex, midway between lambda and bregma sutures. A pneumatic piston impactor device (Biomedical Engineering Facility, Virginia Commonwealth University, Richmond, VA) with a 5mm-diameter, rounded tip was used to create a moderate injury (2.0 mm depth, 4.0 m/s velocity, and 130 ms duration of impact). Following injury, the bone flap was replaced and sealed with bone wax, and the skin was sutured closed with 3.0 black braided silk sutures.
4.2.
Experimental design
Fifty-two rats in CCI injured groups were randomly assigned to receive 0.9% normal saline (NS; n = 12), 7.5% hypertonic saline (HS; n = 15), delayed NS (n = 3), delayed HS (n = 4), or no treatment (CCI control; n = 18). Five rats were uninjured, untreated controls (controls) for brain histological analyses and five rats were used for serum analyses. Fluid treated rats were infused with 4 ml/kg of either 7.5% NaCl or 0.9% NaCl normal saline through the femoral vein (polyethylene 10 catheter) using a syringe infusion pump (model 200; KD Scientific, Holliston, MA) at a rate of 0.2 ml/min. Immediate treatment began 10 min after injury, whereas delayed treatments were administered at 1 h after injury. Percent tissue loss determined using cresyl violet staining and GFAP positive immunoreactivity were quantified in a blinded fashion at 7 days after CCI injury. Statistical comparisons were performed using a one-way analysis of variance (ANOVA). Post hoc between group comparisons were performed using the Tukey's multiple comparison Test. Pearson correlation was used to evaluate the relationship between histological assessments. Significance levels were set at p < 0.05 for all statistical analyses and results are reported as the mean and SEM. All data were analyzed using the Prism statistical program.
4.3.
Histological analyses
Rats were euthanized with a lethal dose of intraperitoneal sodium pentobarbital (1.5 ml), then perfused transcardially with 200 ml normal saline followed by 200 ml ice-cold 4% paraformaldehyde (Sigma) for fixation. Brains were stored in 4% paraformaldehyde overnight at 4 °C, then transferred to a 30% sucrose solution for 3 days for cryoprotection. Brains were then sectioned coronally (20 μm) on a freezing microtome with approximately 400 μm distance between slices. Serial sections in a 1:10 series were used for histological analysis, mounted
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
onto gelatin coated slides and dried overnight. Sections were stained with 0.2% cresyl violet (Sigma Chemical, St. Louis, MO) for general histological examination and measurement of tissue loss. Alternate sections were stained with a monoclonal antibody against glial fibrillary acidic protein (GFAP; clone GA5; Sigma Chemical Co., St. Louis, MO) and biotinylated donkey anti-mouse IgG secondary antibody (Jackson ImmunoResearch) to assess changes in astrocyte morphology indicative of reactivity (hypertrophy and/or proliferation) and quantify changes in the percent area fraction (defined below) and number of GFAP+ immunoreactive cells. GFAP was visualized using DAB (Sigma) and hematoxylin (Gill-1; Sigma) was used as a counterstain. Stained sections were dehydrated in graded alcohol, and defatted in xylene (Fisher Scientific) before being coverslipped with Permount (Fisher Scientific). Cresyl violet stained sections were viewed using light microscopy for gross morphology (Olympus BX5) and photographed using a Nikon Coolpix 950 digital camera at 2× magnification. Percent tissue loss in cresyl violet stained tissue was measured using NIH ImageJ software and reported as the percent volume of tissue loss. The volume of tissue loss was calculated by multiplying the area of the tissue loss delineated by the absence of cresyl violet staining for each section by the thickness of each section, and summing the products. Area of tissue loss was measured by subtracting the area of remaining tissue in the ipsilateral hemisphere from the area of tissue in the contralateral hemisphere. Percent volume of tissue loss was calculated as the volume of tissue loss divided by the volume of the contralateral hemisphere multiplied by 100. Brain tissues were analyzed quantitatively for GFAP+ cells using a Nikon fluorescence microscope interfaced with a quantification system (Bioquant System, Bioquant, TN) and an X,Y motorized stage. Quiescent GFAP+ astrocytes and reactive GFAP+ astrocytes (evidenced by hypertrophied soma and processes and cell proliferation) were captured using A Retiga EXI cooled camera (QImaging, Surrey, BC). GFAP immunoreactivity was systematically quantified in a region of interest that included the sensorimotor cortex immediately adjacent to the injury extending to the sagittal sulcus using an irregular specialty measurement tool available with the image analysis system (Bioquant, Nashville, TN). GFAP+ immunoreactivity was reported as a percent area fraction as further described by Al-Shatti et al. (2005). The mean area fraction of thresholded immunoreactive product in the selected region of interest was determined by dividing the video count area of pixels above background thresholds by the total number of pixels in the entire chosen image field as described previously (Elliott et al., 2008a). Video count area is the number of pixels in an image field that meet a user defined criterion multiplied by the area of a pixel at the selected magnification (20× objective and 350× magnification factor for this analysis). GFAP percent area fraction determines the quantity of GFAP staining (number of pixels above threshold) appearing in a given area whereby reactive astrocytes can be identified, however, does not distinguish between astrocyte hypertrophy and proliferation. Therefore, high-power morphological evaluation and cell counting were also performed to determine whether hypertrophy (defined as increased length and/or girth of GFAP positive soma and processes) or proliferation (defined as increased cell number) was present. An independent random sampling approach described by Mouton (2002) was used to count the number of GFAP+ cells (Mouton, 2002). The mean number of GFAP+ labeled cells was counted in the ipsilateral cortex adjacent to the injury and closest to the midline under 100× magnification in which three measurements were made
189
per section. Every 10th serial section through the injury was counted in order to determine the mean number of GFAP+ cells in a set volume of tissue (0.27 mm3) per 3 sections. To avoid bias in estimating the mean number of GFAP+ labeled cells, only cells in which the nucleus (unlabeled) was visible were measured. All measurements for cresyl violet and GFAP analyses were performed in a blinded manner.
4.4.
Motor behavior assessment
A balance beam test for motor coordination and balance was conducted before and 7 days after CCI injury in all treatment and control groups (Feeney et al., 1982; Goldstein and Davis, 1990; Garcia et al., 1995; Sell et al., 2008). The balance beam (2 × 4 × 122 cm wooden beam at a height of 72 cm) has a rectangular box (32.5 × 20.5 × 18.0 cm) on one end that served as a motivational tool for a place of rest and comfort. Rats learned how to cross the balance beam prior to any scoring of trials usually within three trials. The scoring for the balance beam test ranges from 1 to 7 in which 1 represents the greatest motor deficit and 7 represents normal.
4.5.
Serum analysis
Sprague–Dawley Rats (n = 5) were anesthetized with xylazine and ketamine (1:1) and followed by cannulation of both the right and left femoral veins for fluid infusion and blood withdraw, respectively. Blood samples were collected with a syringe flushed with heparin at baseline prior to hypertonic saline infusion and following infusion of a single 4 ml/kg bolus of 7.5% HS. Serum was collected at 10 min and 2, 3, 4, and 24 h after infusion completion. Plasma osmolarity and sodium concentrations were measured using a Nova Blood Gas Analyzer.
Acknowledgments Support for this research was provided by the PA Department of Health and AHA (0415405U).
REFERENCES
Al-Shatti, T., Barr, A.E., Safadi, F.F., Amin, M., Barbe, M.F., 2005. Increase in inflammatory cytokines in median nerves in a rat model of repetitive motion injury. J. Neuroimmunol. 167, 13–22. Anderson, J.T., Wisner, D.H., Sullivan, P.E., Matteucci, M., Freshman, S., Hildreth, J., Wagner Jr., F.C., 1997. Initial small-volume hypertonic resuscitation of shock and brain injury: short- and long-term effects. J. Trauma 42, 592–600 discussion 600-591. Baskaya, M.K., Dogan, A., Temiz, C., Dempsey, R.J., 2000. Application of 2,3,5-triphenyltetrazolium chloride staining to evaluate injury volume after controlled cortical impact brain injury: role of brain edema in evolution of injury volume. J. Neurotrauma 17, 93–99. Boycott, H.E., Wilkinson, J.A., Boyle, J.P., Pearson, H.A., Peers, C., 2008. Differential involvement of TNF alpha in hypoxic suppression of astrocyte glutamate transporters. Glia 56, 998–1004. Chang, Y.C., Kim, H.W., Rapoport, S.I., Rao, J.S., 2008. Chronic NMDA administration increases neuroinflammatory markers
190
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 –19 1
in rat frontal cortex: cross-talk between excitotoxicity and neuroinflammation. Neurochem. Res. 33, 2318–2323. Chen, Y., Swanson, R.A., 2003. Astrocytes and brain injury. J. Cereb. Blood. Flow. Metab. 23, 137–149. Chen, S., Pickard, J.D., Harris, N.G., 2003. Time course of cellular pathology after controlled cortical impact injury. Exp. Neurol. 182, 87–102. Doyle, J.A., Davis, D.P., Hoyt, D.B., 2001. The use of hypertonic saline in the treatment of traumatic brain injury. J. Trauma Inj. Infect. Crit. Care 50, 367–383. Elliott, M.B., Jallo, J.J., Gaughan, J.P., Tuma, R.F., 2007. Effects of crystalloid–colloid solutions on traumatic brain injury. J. Neurotrauma 24, 195–202. Elliott, M.B., Barr, A.E., Kietrys, D.M., Al-Shatti, T., Amin, M., Barbe, M.F., 2008a. Peripheral neuritis and increased spinal cord neurochemicals are induced in a model of repetitive motion injury with low force and repetition exposure. Brain. Res. 1218, 103–113. Elliott, M.B., Jallo, J.J., Tuma, R.F., 2008b. An investigation of cerebral edema and injury volume assessments for controlled cortical impact injury. J. Neurosci. Methods 168, 320–324. Feeney, D.M., Gonzalez, A., Law, W.A., 1982. Amphetamine, haloperidol, and experience interact to affect rate of recovery after motor cortex injury. Science 217, 855–857. Garcia, J.H., Wagner, S., Liu, K.F., Hu, X.J., 1995. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats. Statistical validation. Stroke 26, 627–634 discussion 635. Ghirnikar, R.S., Lee, Y.L., Eng, L.F., 1998. Inflammation in traumatic brain injury: role of cytokines and chemokines. Neurochem. Res. 23, 329–340. Goldstein, L.B., Davis, J.N., 1990. Beam-walking in rats: studies towards developing an animal model of functional recovery after brain injury. J. Neurosci. Methods 31, 101–107. Hampton, D.W., Rhodes, K.E., Zhao, C., Franklin, R.J., Fawcett, J.W., 2004. The responses of oligodendrocyte precursor cells, astrocytes and microglia to a cortical stab injury, in the brain. Neuroscience 127, 813–820. Harutjunyan, L., Holz, C., Rieger, A., Menzel, M., Grond, S., Soukup, J., 2005. Efficiency of 7.2% hypertonic saline hydroxyethyl starch 200/0.5 versus mannitol 15% in the treatment of increased intracranial pressure in neurosurgical patients—a randomized clinical trial [ISRCTN62699180]. Crit. Care 9, R530–R540. Horn, P., Munch, E., Vajkoczy, P., Herrmann, P., Quintel, M., Schilling, L., Schmiedek, P., Schurer, L., 1999. Hypertonic saline solution for control of elevated intracranial pressure in patients with exhausted response to mannitol and barbiturates. Neurol. Res. 21, 758–764. Junger, W.G., Hoyt, D.B., Davis, R.E., Herdon-Remelius, C., Namiki, S., Junger, H., Loomis, W., Altman, A., 1998. Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38. J. Clin. Invest. 101, 2768–2779. Khanna, S., Davis, D., Peterson, B., Fisher, B., Tung, H., O'Quigley, J., Deutsch, R., 2000. Use of hypertonic saline in the treatment of severe refractory posttraumatic intracranial hypertension in pediatric traumatic brain injury. Crit. Care Med. 28, 1144–1151. Knapp, J.M., 2005. Hyperosmolar therapy in the treatment of severe head injury in children: mannitol and hypertonic saline. AACN Clin. Issues 16, 199–211. Langlois, J.A., Rutland-Brown, W., Wald, M.M., 2006. The epidemiology and impact of traumatic brain injury: a brief overview. J. Head Trauma Rehabil. 21, 375–378. Levene, H.B., Erb, C.J., Gaughan, J.P., Loftus, C.M., Tuma, R.F., Jallo, J.I., 2007. Hypertonic saline as a treatment for acute spinal cord injury: effects on somatic and autonomic outcomes as observed in a mouse model. Clin. Neurosurg. 54, 213–219.
Mouton, P.R. (Ed.), 2002. Principles and practices of unbiased stereology. The Johns Hopkins University Press, Baltimore. Munar, F., Ferrer, A.M., de Nadal, M., Poca, M.A., Pedraza, S., Sahuquillo, J., Garnacho, A., 2000. Cerebral hemodynamic effects of 7.2% hypertonic saline in patients with head injury and raised intracranial pressure. J. Neurotrauma 17, 41–51. Myer, D.J., Gurkoff, G.G., Lee, S.M., Hovda, D.A., Sofroniew, M.V., 2006. Essential protective roles of reactive astrocytes in traumatic brain injury. Brain 129, 2761–2772. Okuno, S., Nakase, H., Sakaki, T., 2001. Comparative study of 2,3, 5-triphenyltetrazolium chloride (TTC) and hematoxylin–eosin staining for quantification of early brain ischemic injury in cats. Neurol. Res. 23, 657–661. Otani, N., Nawashiro, H., Fukui, S., Ooigawa, H., Ohsumi, A., Toyooka, T., Shima, K., Gomi, H., Brenner, M., 2006. Enhanced hippocampal neurodegeneration after traumatic or kainate excitotoxicity in GFAP-null mice. J. Clin. Neurosci. 13, 934–938. Pascual, J.L., Ferri, L.E., Seely, A.J., Campisi, G., Chaudhury, P., Giannias, B., Evans, D.C., Razek, T., Michel, R.P., Christou, N.V., 2002. Hypertonic saline resuscitation of hemorrhagic shock diminishes neutrophil rolling and adherence to endothelium and reduces in vivo vascular leakage. Ann. Surg. 236, 634–642. Phillis, J.W., Song, D., O'Regan, M.H., 1999. Effects of hyperosmolarity and ion substitutions on amino acid efflux from the ischemic rat cerebral cortex. Brain Res. 828, 1–11. Prow, N.A., Irani, D.N., 2008. The inflammatory cytokine, interleukin-1 beta, mediates loss of astroglial glutamate transport and drives excitotoxic motor neuron injury in the spinal cord during acute viral encephalomyelitis. J. Neurochem. 105, 1276–1286. Qureshi, A.I., Suarez, J.I., 2000. Use of hypertonic saline solutions in treatment of cerebral edema and intracranial hypertension. Crit. Care Med. 28, 3301–3310. Qureshi, A.I., Suri, M.F., Ringer, A.J., Guterman, L.R., Hopkins, L.N., 2002. Regional intraparenchymal pressure differences in experimental intracerebral hemorrhage: effect of hypertonic saline. Crit. Care. Med. 30, 435–441. Rotstein, O.D., 2000. Novel strategies for immunomodulation after trauma: revisiting hypertonic saline as a resuscitation strategy for hemorrhagic shock. J. Trauma Inj. Infect. Crit. Care 49, 580–583. Saatman, K.E., Duhaime, A.C., Bullock, R., Maas, A.I., Valadka, A., Manley, G.T., 2008. Classification of traumatic brain injury for targeted therapies. J. Neurotrauma 25, 719–738. Sama, M.A., Mathis, D.M., Furman, J.L., Abdul, H.M., Artiushin, I.A., Kraner, S.D., Norris, C.M., 2008. Interleukin-1beta-dependent signaling between astrocytes and neurons depends critically on astrocytic calcineurin/NFAT activity. J. Biol. Chem. 283, 21953–21964. Sell, S.L., Avila, M.A., Yu, G., Vergara, L., Prough, D.S., Grady, J.J., DeWitt, D.S., 2008. Hypertonic resuscitation improves neuronal and behavioral outcomes after traumatic brain injury plus hemorrhage. Anesthesiology 108, 873–881. Shackford, S.R., 1997. Effect of small-volume resuscitation on intracranial pressure and related cerebral variables. J. Trauma Inj. Infect. Crit. Care 42, 48S–53S. Shackford, S.R., Bourguignon, P.R., Wald, S.L., Rogers, F.B., Osler, T.M., Clark, D.E., 1998. Hypertonic saline resuscitation of patients with head injury: a prospective, randomized clinical trial. J. Trauma Inj. Infect. Crit. Care 44, 50–58. Simma, B., Burger, R., Falk, M., Sacher, P., Fanconi, S., 1998. A prospective, randomized, and controlled study of fluid management in children with severe head injury: lactated Ringer's solution versus hypertonic saline. Crit. Care Med. 26, 1265–1270. Soustiel, J.F., Vlodavsky, E., Zaaroor, M., 2006. Relative effects of mannitol and hypertonic saline on calpain activity, apoptosis and polymorphonuclear infiltration in traumatic focal brain injury. Brain Res. 1101, 136–144.
BR A I N R ES E A RC H 1 3 0 5 ( 2 00 9 ) 1 8 3 – 1 91
Spera, P.A., Arfors, K.E., Vasthare, U.S., Tuma, R.F., Young, W.F., 1998. Effect of hypertonic saline on leukocyte activity after spinal cord injury. Spine 23, 2444–2448 discussion 2448-2449. Spera, P.A., Vasthare, U.S., Tuma, R.F., Young, W.F., 2000. The effects of hypertonic saline on spinal cord blood flow following compression injury. Acta Neurochir. (Wien) 142, 811–817. Sumas, M.E., Legos, J.J., Nathan, D., Lamperti, A.A., Tuma, R.F., Young, W.F., 2001. Tonicity of resuscitative fluids influences outcome after spinal cord injury. Neurosurgery 48, 167–172 discussion 172-163. Thomale, U., Griebenow, M., Kroppenstedt, S., Unterberg, A.W., Strover, J.F., 2004. Small volume resuscitation with hyperhaes improves pericontusional perfusion and reduces lesion volume following controlled cortical impact injury in rats. J. Neurotrauma 21, 1737–1746. Tuma, R.F., Vasthare, U.S., Arfors, K.E., Young, W.F., 1997. Hypertonic saline administration attenuates spinal cord injury. J. Trauma 42, S54–60. Vialet, R., Albanese, J., Thomachot, L., Antonini, F., Bourgouin, A., Alliez, B., Martin, C., 2003. Isovolume hypertonic solutes (sodium chloride or mannitol) in the treatment of refractory
191
posttraumatic intracranial hypertension: 2 mL/kg 7.5% saline is more effective than 2 mL/kg 20% mannitol. Crit. Care Med. 31, 1683–1687. Ware, M.L., Nemani, V.M., Meeker, M., Lee, C., Morabito, D.J., Manley, G.T., 2005. Effects of 23.4% sodium chloride solution in reducing intracranial pressure in patients with traumatic brain injury: a preliminary study. Neurosurgery 57, 727–736 discussion 727-736. Wenham, T.N., Hormis, A.P., Andrzejowski, J.C., 2008. Hypertonic saline after traumatic brain injury in UK neuro-critical care practice. Anaesthesia 63, 558–559. White, R.E., Jakeman, L.B., 2008. Don't fence me in: harnessing the beneficial roles of astrocytes for spinal cord repair. Restor. Neurol. Neurosci. 26, 197–214. Yilmaz, N., Dulger, H., Kiymaz, N., Yilmaz, C., Gudu, B.O., Demir, I., 2007. Activity of mannitol and hypertonic saline therapy on the oxidant and antioxidant system during the acute term after traumatic brain injury in the rats. Brain Res. 1164, 132–135. Ziai, W.C., Toung, T.J., Bhardwaj, A., 2007. Hypertonic saline: first-line therapy for cerebral edema? J. Neurol. Sci. 261, 157–166.