March 25, 2011 Ms. Mary Ray New Jersey Commission on Brain Injury Research P.O. Box 360 5th Floor, room 502 Trenton, New Jersey 08625 Re: Final Report - NJCBIR Grant #09-3206-BIR-E Role of Bone Marrow cells in Repair of the Blood Brain Barrier after Injury John Glod, MD, PhD, PI Dear Ms. Ray, Attached please find an original and five copies of my final report for the above referenced grant. I would like to thank the New Jersey Commission on Brain Injury Research for providing funding to support this important research. Should you have any questions, please do not hesitate to contact me at 732-235-8864 or glodjo@umdnj.edu Thank you again. Sincerely,

John Glod, MD, PhD Assistant Professor of Pediatrics UMDNJ-Robert Wood Johnson Medical Schol Attachments: original and five copies

COVER PAGE

1. Program Director’s Name, Address, Telephone Number: John Glod, MD, PHD

Cancer Institute of New Jersey Division of Pediatric Hematology/Oncology 195 Little Albany Street New Brunswick, NJ 08903 732-235-8864

2. Name of Institution:

UMDNJ-Robert Wood Johnson Medical School 3. Grant Title:

Role of Bone Marrow cells in Repair of the Blood Brain Barrier after Injury 4. Grant Number:

09-3206-BIR-E-2 5. Grant Period Covered by the Report:

6/1/2008 – 5/31/2010; No cost extension approved to 12/31/2010 6. Date of Submission of Report:

3/25/2011

John Glod, MD, PhD Grant # 09-3206-BIR-E-2 Role of Bone Marrow cells in Repair of the Blood Brain Barrier after Injury

BODY OF REPORT

I. Original aims of the project:

The repair of damaged vasculature is an important component of the pathophysiology of traumatic brain injury. Recent evidence suggests that bone marrow cells such as macrophages and mesenchymal stromal cells (MSCs) are critical for this process. Mesenchymal stromal cells localize to areas of injury in the brain as well as tumors within the central nervous system. Evidence from other laboratories show that the bone marrow is a source of pericytes and our preliminary data demonstrate that glial factors induce the expression of pericyte-like features by MSCs, suggesting that they are the precursors to pericytes. We hypothesize that: 1) MSCs are a source of pericytes during vascular repair in the brain and 2) Macrophages present at a site of brain injury are required for effective recruitment of MSCs and their incorporation into repairing vasculature. In order to test this hypothesis we have pursued the following specific aims: Specific Aim #1: Determine whether MSCs adopt a pericyte phenotype in response to the microenvironment at a site of brain injury.

Specific Aim #2: Define the role of macrophages in recruitment of MSCs to the site of brain injury. 2. Project Successes We have submitted a manuscript for publication. The manuscript is currently in revision. We have demonstrated that macrophages are important in the reformation of the blood brain barrier following injury. Our work shows that blood vessels are less abundant following injury in the context of macrophage depletion. Additionally, experiments suggest that very large diameter vessels may be less impacted in the setting of macrophage depletion. We have shown that human MSCs show in vitro migration in response to factors produced by macrophages in both a cell line model (U937) and primary culture human macrophages, identified soluble factors that are involved in macrophage-induced MSC migration, and identified the JNK signaling pathway as an important component in macrophage / MSC interactions. We have also begun to identify changes in MSC expression of cytokines and cytokine receptors in response to soluble factors produced by macrophages.

John Glod, MD, PhD Grant # 09-3206-BIR-E-2 Role of Bone Marrow cells in Repair of the Blood Brain Barrier after Injury 3. Project Challenges We have not identified significant changes in pericyte phenotype related to macrophage depletion. We have also not demonstrated conclusively that mesenchymal stromal cells develop into pericytes following injury. 4. Implications for future research and/or clinical treatment The requirement for macrophages for optimal repair of the BBB following injury has important clinical implications. Treatments that increase macrophage mobilization such as administration of hematopoietic growth factors may have the potential of improving recover following traumatic brain injury. This work also suggests that factors that impair macrophage production such as infection may lead to increased tissue repair following brain injury. 5. Plans to continue the research, including applications submitted to other sources for ongoing support. We are continuing with work looking at the interaction between macrophages and mesenchymal stromal cells at the site of injury. We further plan to better characterize changes in the time course and architecture of vascular remodeling after traumatic brain injury and better defining the role of macrophages in this process. We plan to use the work funded by the NJCBIR as the basis for an NIH grant application. 6. Explain how you have leveraged NJCBIR funding to obtain additional federal or other support for brain injury research and list the appropriate funding organizations. NJCBIR funding has been used to generate data that will form the basis of planned applications for federal NIH funding. 7. List and including a copy of all publications emerging from this research including those used in preparation. Macrophages play a key role in early blood brain barrier reformation after hypothermic brain injury. Koneru, R, Kobiler, D., Lehrer, S., Li, J, van Rooijen, N., Banerjee, D., and Glod, J. Manuscript in revision.

John Glod, MD, PhD Grant # 09-3206-BIR-E-2 Role of Bone Marrow cells in Repair of the Blood Brain Barrier after Injury See attached 8. Financial Summary. Final Financial Report was submitted on 1/13/11 by the UMDNJ Grants Office – see attached.

John Glod, MD, PhD Grant # 09-3206-BIR-E-2 Role of Bone Marrow cells in Repair of the Blood Brain Barrier after Injury

PUBLICATIONS AND PRESENTATIONS All papers, presentations, chapter, and abstracts should mention that the research was supported by a grant from the New Jersey Commission on Brain Injury Research. Copies must be sent to the NJCBIR office. Copies must be sent to the NJCBIR office, even if you inadvertently forgot to cite NJCBIR support. See attached.

Macrophages Play a Key Role in early Blood Brain Barrier Reformation after

Hypothermic Brain Injury

Rajeth Koneru3, David Kobiler3,5, Shoshona Lehrer3,5, Jiadong Li3, Nico van Rooijen4,

Debabrata Banerjee1,2 , and John Glod2,3

Departments of 1Medicine, 2Pharmacology and 3Pediatrics, The Cancer Institute of New Jersey. Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey; 4Department of Molecular Cell Biology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands. 5Current address IIBR, Ness Ziona, Israel.

Key words: blood brain barrier (BBB); macrophage

Running title: Macrophage depletion delays BBB reformation after injury

Correspondence: John Glod, M.D., Ph.D., glodjo@umdnj.edu; The Cancer Institute of

New Jersey, RWJMS/UMDNJ, 195 Little Albany street, New Brunswick, New Jersey

08903, USA. Telephone: 732-235-9854; Fax: 732-235-8234

Abstract

The repair of damaged vasculature is an important component of the pathophysiology of

traumatic brain injury. In addition to re-establishing perfusion to areas supplied by damaged

vessels, the integrity of the blood brain barrier (BBB) must be reformed at sites of injury. Recent

evidence suggests that bone marrow-derived cells such as macrophages are critical for this

process. Cells of the monocyte/macrophage lineage may play a role in formation of the BBB

through modulation of the phenotype of microvascular endothelial cells or potentially through a

structural role in the vasculature We have used liposomal clodronate to deplete monocytes and

tissue macrophages. This method led to a marked reduction in the accumulation of F4/80-

expressing cells at sites of hypothermic brain injury in a murine model. The reduction in

macrophages at the injury site was accompanied by a decrease in neovascularization following

the injury. We also evaluated the reformation of the blood brain barrier after injury. In control

animals the permeability of the BBB at the injury to FITC-labeled albumin returned to normal

levels by seven days post-injury. In macrophage-depleted mice leakage of albumin was still

observed at seven days post-injury. These results suggest that macrophages play an important

role in both post-traumatic revascularization as well as early repair of the BBB after injury.

Introduction

A critical component of tissue repair following traumatic brain injury (TBI) is the

reformation of the brain vasculature. Evidence suggests that reformation of the vascular network

following traumatic injury to the CNS is important for functional recovery. Inhibition of

angiogenesis after TBI in a rodent model through vascular endothelial growth factor (VEGF)

blockade led to increases in the area of hemorrhage and increases in serum markers of neuronal

injury as well as a decrease in vascular density (Skold et al., 2006). Conversely, increased

angiogenesis following experimental CNS injury after treatment with atorvastatin or VEGF led to

improved functional recovery in a TBI model (Lu et al., 2004) and decreased secondary tissue

degeneration in a spinal cord contusion model (Widenfalk et al., 2003). Another important

component to vascular repair in the CNS is restoration of blood brain barrier (BBB) integrity.

The BBB is a complex system contributed to by endothelial cells, astroglia, perivascular

macrophages, and pericytes that regulates the movement of substances from the circulation to the

brain parenchyma and vice versa (Abbott, 2000; Kniesel and Wolburg, 2000; Lee et al., 2009;

Rubin and Staddon, 1999). Impaired BBB function is seen after brain injury and is related to

neuronal injury (Dinapoli et al., 2007; Ping et al., 2005). In addition to TBI, impaired BBB

function has been implicated in the pathophysiology of diseases including Alzheimer disease,

multiple sclerosis and CNS infection (Adams et al., 2002; Annunziata, 2003; Floris et al., 2004;

Jellinger, 2002; Ujiie et al., 2003). Thus, outcome after brain injury may be impacted by both

revascularization as well as reformation of the blood brain barrier.

Cells of the monocyte / macrophage lineage play an important role in blood vessel

growth. An increase in tissue macrophages accompanies angiogenesis after

stroke(Manoonkitiwongsa et al., 2001) and VEGF mediated angiogenesis in the brain(Croll et al.,

2004) and depletion of peripheral blood monocytes inhibits vascularization of the choroid of the

eye (Espinosa-Heidmann et al., 2003; Sakurai et al., 2003) and collateral artery growth in a limb

ischemia model(Heil et al., 2002). Macrophages may perform multiple functions during

angiogenesis including production of pro-angiogenic factors such as VEGF and placenta-derived

growth factor (PDGF) and disruption of the extracellular matrix during vessel growth (Fujiyama

et al., 2003; Moldovan et al., 2000). Populations of monocytes and macrophages may also play a

structural role in new blood vessels. Myeloid cells have been reported to assume endothelial

characteristics (Fernandez Pujol et al., 2000; Fernandez Pujol et al., 2001; Glod et al., 2006;

Harraz et al., 2001; Havemann et al., 2003; Nakul-Aquaronne et al., 2003; Rehman et al., 2003;

Schmeisser et al., 2001) and perivascular macrophages contribute to both the blood-retinal and

blood-nerve barriers(Gray et al., 2007; Mendes-Jorge et al., 2009).

We sought to determine the importance of macrophages in repair of brain vasculature

following traumatic brain injury. Treatment of mice with liposomal clodronate led to decreased

peripheral blood monocytes and a marked reduction in the accumulation of F4/80 expressing cells

at sites of hypothermic brain injury. Macrophage depletion led to decreased vascularization

following injury and also a delay in the reconstitution of the blood brain barrier as measured by

FITC-albumin leakage. These results suggest that macrophages play an important role in both

vascular growth and reconstitution of the BBB following traumatic brain injury.

Methods

Animal Studies/Injury Model

Three to four week old female Swiss-Webster mice were purchased from Taconic Farms and

utilized in the brain injury model. The experimental group was treated with liposomal

clodronate, a monocyte depleting agent(van Rooijen et al., 1996). Clodronate (a gift from Roche

Diagnostics 120 GmbH, Mannheim, Germany) was encapsulated into liposomes as described

previously(Van Rooijen and Sanders, 1994). Mice were injected via tail vein with 100μl of either

liposomal clodronate or PBS liposomes 3-4 days prior to hypothermic injury, on the day of

injury, and every 3-4 days thereafter until the time of sacrifice. Hypothermic brain injury was

performed as previously described (Nag, 1996). Animals were deeply anesthetized and placed in

a David Kopf stereotaxic apparatus. The skin surface was cleansed with 100% ethanol. A small

incision was made in the skin of the skull. The coordinates of the bregma were noted and the

injury was located 1mm anterior and 1.5 mm lateral of bregma. A cold stainless steel 2 mm

diameter cylindrical rod was set in liquid nitrogen for a few minutes. After removal from liquid

nitrogen the rod was immediately placed on the injury site for 60 seconds. The site was then

cleansed with betadine and the skin was closed with a surgical staple. All animal procedures

were approved by the Animal Care and Use Committee of RWJMS.

Vessel Quantitation

On the day of sacrifice mice were deeply anesthetized and perfused with phosphate

buffered saline (PBS) through the left ventricle. Brains were removed, placed in a dry-ice

cooled solution of isopentane for 10 seconds, and then placed in a cryomold on dry-ice in the

proper orientation for coronal tissue sectioning. The mold was filled with Tissue-Tek O.C.T.

compound (Sakura Finetek). After the O.C.T. solidified, the tissue was placed in a Lecia

cryostat for 1 hour at -20 degrees. Ten micron sections were cut and adhered to microscope

slides and placed in methanol at 4 degrees for 10 minutes then washed in cold PBS. Tissue

sections were then stained for expression of vonWillebrand factor to label blood vessels.

All tissue sections were imaged using a Nikon Eclipse C1 Confocal Microscope. Images

were obtained using a 60X oil lens. Vessel quantitation was performed using Adobe Photoshop

CS3 software. Each injury site was divided into fields of 3000 pixels X 1800 pixels. Vasculature

was quantitated in sections at the injury site as well as sections of uninjured cortex. The number

of pixels that were labeled for von Willebrand factor (VWF) was determined for each field using

a magic wand tool. The von Willebrand staining per field was averaged across a minimum of

seven fields for each animal.

Immunoflourscence

The following primary antibodies were used in these experiments: anti-human von Willebrand

Factor (Dako), anti-mouse F4/80 (Abcam), and rabbit anti-GFAP (Sigma). Labeled goat anti-

rabbit and anti-rat secondary antibodies were purchased from Molecular Probes. Slides were

immersed in a dish containing blocking buffer (consisted of 10% goat serum (Dako) + 0.01%

Triton X-100 (Sigma) in PBS) for 1 hour in a humidified chamber at 37 degrees. The tissue

sections were covered with primary antibody diluted in blocking buffer. After incubation for 1

hour at 37°C in humidified chamber, excess liquid was blotted from slides and then rinsed three

times in PBS for five minutes per wash. Secondary antibody labeling was performed in a similar

manner. Slides were mounted in Vectashield with DAPI (Vector Labs). All tissue sections were

imaged using a Nikon Eclipse C1 Confocal Microscope. Images were obtained using a 60X oil

lens.

BBB leakage

Swiss-Webster mice (female) approx 3-4 weeks old were purchased from Taconic Farms and

utilized in Blood Brain Barrier leakage study. Mice were injected through the tail vein with

either liposomal clodronate or liposomes containing PBS 3-4 days prior to cold shock injury, on

the day of injury, and every 3-4 days thereafter until the time of sacrifice. Hypothermic brain

injury was performed as described above. On the day of sacrifice mice were injected i.p. with an

anesthetic (Ketaset(100mg/ml) /Acepromazine(1:2)). Thirty minutes prior to the time of sacrifice

mice were injected via tail vein with 100μl of Sodium Flourescein labeled albumin (2.5% in

PBS). Five minutes prior to the time of sacrifice mice were injected via tail vein with 100μl of

Rhodamine Dextran (2.5% w/v in PBS). At the time of sacrifice, mice were decapitated and the

brain hemispheres were removed, weighed and stored in separate tubes on dry ice. The tissue

was homogenized and centrifuged. The supernatant was collected and the concentration of

Fluorescien albumin and rhodamine dextran in each hemisphere were determined using a

fluorimeter. The concentrations of Rhodamine dextran and FITC-albumin in the uninjured

hemisphere were used to calculate the amount of FITC-albumin present in the vasculature. The

intravascular FITC-albumin was then subtracted from the total FITC-albumin in the injured

hemisphere to calculate the amount of leakage.

Amount of leakage = Alb(inj) – (Alb(con)*Rhod(inj)/Rhod(con))

Results

Treatment with liposomal clodronate results in decreased accumulation of F4/80 expressing cells

after brain injury.

Swiss-Webster mice were treated with liposomal clodronate every four days beginning four days

prior to injury. Animals were sacrificed seven days post-injury. Hematoxylin and eosin staining

of tissue sections through the injury site demonstrated an overall decrease in cellularity in animals

treated with liposomal clodronate (Figure 1a). More specifically, immunohistochemical staining

for the macrophage marker F4/80 showed a decrease in the accumulation of macrophages at the

injury site in animals that were treated with liposomal clodronate (Figure 1b). Tissue sections

through injury sites were also stained for glial fibrillary acidic protein. Both control animals as

well as mice treated with liposomal clodronate had a brisk gliosis present at day 7 after injury

(Figure 1c).

Macrophage depletion decreases the angiogenic response following cold injury.

Angiogenesis following injury was quantitated. Tissue sections from control animals and

animals treated with liposomal clodronate were stained for the endothelial protein von Willebrand

factor. In preliminary experiments vessel density following hypothermic injury increased until

day seven following injury and then plateaued (Figure 2a). Other investigators have reported a

similar time-course of vascularization following CNS injury (Nag, 2002). Revascularization

following injury was then compared between animals treated with clodronate and controls. A

decrease in vessel density between control animals and macrophage-depleted animals was evident

at day 7 after injury and persisted throughout the length of the experiment (Figure 2b and 2c).

Animals treated with liposomal clodronate had an approximately 45% decrease in von Willebrand

factor staining at day 7 after injury (5392 +/- 3113 pixels per field compared to 9890 +/- 4161

pixels per field; p=0.0147) (Figure 2b). There was no significant difference in the vascular

density between liposomal clodronate treated and control mice in an area of normal brain adjacent

to the injury site (2267+/-1718 pixels per field compared to 3058+/-1924 pixels; p=.0967) (B).

At day fourteen mice treated with liposomal clodronate had an area of staining of 7866 +/- 872

pixels per field compared to 11918+/- 1052 pixels per field for control mice (p = 0.003). Again

there was no significant difference in the vascular density in areas of normal brain adjacent to the

injury site (6621+/-289 pixels per field compared to 5191+/-1448 pixels per field; p=0.15) (n= 5

for mice treated with liposomal clodronate and n=3 for control mice) (C).

Macrophage depletion causes a delay in blood brain barrier repair after injury

In order to assess the role of macrophages in BBB repair, the leakage of FITC-labeled

albumin was measured after injury. In control mice albumin leakage was seen at 3 days after

injury, but by 7 days after injury the degree of albumin extravasation at the injury site was

indistinguishable form the contra lateral hemisphere (Figure 3). This rate of reformation of the

BBB after injury is similar to results seen by others (Nag, 2002). In mice that were treated with

liposomal clodronate albumin leakage continued through day 7 after injury. At day three

following injury the leakage of FITC-albumin was the same in both the liposomal clodronate

treated and control animals (301 +/- 126 compared to 259 +/- 153; p=0.7). A non-significant

trend toward increased FITC-albumin leak was seen in the liposomal clodronate treated animals

at day 5. At seven days after injury there was continued leakage of FITC-albumin in the

clodronate treated animals and minimal leakage in the control animals (314 +/- 51 compared to

22 +/- 70; p=0.005). These data indicate that the presence of macrophages is important for

reformation of the BBB following injury.

Discussion

Angiogenesis and reformation of the blood brain barrier play a critical role in the re-

establishment of homeostasis in the central nervous system following traumatic injury. While the

role of macrophages in blood vessel formation has been described for a number of organ systems,

the impact of macrophages on the reformation of the specialized vasculature of the brain is not

completely understood. A relationship between macrophage accumulation and cerebrovascular

angiogenesis following injury has been described by several groups and accumulation of

macrophages coincides with changes in microvessel density following focal cerebral ischemia as

well as cold injury in animal models (Manoonkitiwongsa et al., 2001; Nourhaghighi et al., 2003).

Macrophages are likely to have a variety of important functions during this process including the

“Clean-up” of necrotic debris(Manoonkitiwongsa et al., 2001) and regulation of blood vessel

growth and barrier integrity through the elaboration of factors such as angiopoietin-1 and

angiopoietin-2 (Nourhaghighi et al., 2003). Here we provide direct evidence that macrophages

are required for vascular proliferation following injury by demonstrating that macrophage

depletion impairs the formation of new blood vessels. Additionally, we demonstrate directly that

decreased levels of macrophage infiltration impairs another critical component of vascular repair

in the central nervous system, the reformation of the blood brain barrier.

Our data show that depletion of macrophage accumulation at an injury site perturbs the

increase in vascular density that is seen following brain injury. Macrophages are likely to

promote angiogenesis through a number of mechanisms. Macrophages secrete important pro-

angiogenic cytokines such as IL-8 (Carmi et al., 2009) and IL-1 (Hong et al., 2009) and enhance

degradation and reformation of the extracellular matrix through the production of matrix

metalloproteinases (Giraudo et al., 2004; Johnson et al., 2004). A more precise understanding of

the exact role of macrophages in these critical aspects of brain angiogenesis is complicated and

awaits further study.

Our data also provide evidence for an important role for macrophages in reformation of

the blood brain barrier. Brain macrophages have been implicated previously in maintenance of

BBB integrity. Using in vitro co-culture systems it has been reported that peripheral blood

macrophages enhance the tightness of a BBB model (Glod et al., 2006; Zenker et al., 2003) and

perivascular microglial cells within the CNS have been shown to participate in the CNS vascular

through the uptake of macromolecules(Mato et al., 1996). Others have reported that perivascular

macrophages migrate to areas of vascular leak and participate in barrier formation in the blood

retinal barrier (Mendes-Jorge et al., 2009) and the area postrema (Willis et al., 2007).

Additionally, macrophage infiltration into the CNS in an experimental autoimmune

encephalomyelitis model was associated with areas with a more intact BBB (Ladewig et al.,

2009) suggesting that macrophages may be associated with local BBB repair.

Interestingly, the degree of angiogenesis, macrophage infiltration, and the degree of BBB

dysfunction have all been related to neuronal damage or functional recovery after injury.

However, the relationship between these processes and recovery are complicated. While

angiogenic agents such as vascular endothelial growth factor have been reported to have

neuroprotective effects (Carmeliet and Storkebaum, 2002; Ferrara and Gerber, 2001; Sun et al.,

2003), recent information suggests that increased angiogenesis facilitated by the administration of

VEGF may be accompanied by increased macrophage infiltration as well as neuronal

damage(Manoonkitiwongsa et al., 2006). A subset of macrophages have been shown to play an

important anti-inflammatory role following spinal cord injury(Shechter et al., 2009). It is

possible that subtle changes in the inflammatory response have a significant impact on the extent

and timing of revascularization and reinstitution of the blood brain barrier following traumatic

injury.

In summary, macrophages play an extensive role in vascular repair in the brain after

injury. Their impact is not confined to elaboration of pro-angiogenic factors but also includes

roles in the regulation of the vascular architecture and BBB integrity. A better understanding of

the interactions between macrophages and the repairing CNS vasculature could identify

therapeutic targets for improving recovery after brain injury.

Acknowledgment

This work was supported by the New Jersey Commission on Brain Injury; grant

No.08-3206-BIR-E-1 to J.G.

Author Disclosure Statement

No competing financial interests exist.

Figure Legends

Figure 1: Treatment with liposomal clodronate leads to decreased accumulation of

macrophages and impaired resolution of necrosis following hypothermic brain injury.

Mice were treated with liposomal clodronate of phosphate buffered saline before and

after hypothermic brain injury. Seven days after injury the animals were sacrificed and

tissue sections were taken through the injury sites. H & E staining shows a decrease in

the level of cellularity at the injury site of animals treated with clodronate as well as an

accumulation of necrotic debris compared to controls (A). Staining for the macrophage

marker F4/80 demonstrates a marked decrease in the accumulation of macrophages at the

injury site in animals that were treated with liposomal clodronate compared to control

animals (B). Additionally, immunohistochemical staining for glial fibrillary acidic

protein shows brisk gliosis after injury in both the treated and untreated animals(C).

Figure 2: Depletion of peripheral blood monocytes leads to delayed neovascularization

after injury. Mice were subjected to hypothermic brain injury and the time course of

neovascularization following injury was determined through the quantitation of von

Willibrand expressing vessels at the injury site. The number of vessels per field was

counted for a minimum of seven fields at each time point. (A) The majority of

neovascularization occurs by day seven after injury. There was no increase in von

Willibrand Factor staining at the injury site at 3 weeks after injury compared to seven

days after injury. The impact of macrophage depletion with liposomal clodronate on

revascularization following injury was then determined. Tissue sections through the

injury site at day seven and 14 were stained for von Willibrand Factor. The total pixels

per field exhibiting staining for vWF at each injury site was quantitated. At day seven

mice treated with liposomal clodronate had a decreased area of staining compared to

control mice treated with liposomal PBS (5392 +/- 3113 pixels compared to 9890 +/-

4161 pixels; p = 0.0147) (B). There was no significant difference in the vascular density

between liposomal clodronate treated and control mice in an area of normal brain

adjacent to the injury site (2267+/-1718 pixels per field compared to 3058+/-1924 pixels;

p=.0967) ( n=3 for liposomal clodronate treated mice and n=3 for control mice) (B). The

difference in vascular density at the injury site between liposomal clodronate treated mice

and control mice was less pronounced 14 days after injury. At day fourteen mice treated

with liposomal clodronate had an area of staining of 7866 +/- 872 pixels per field

compared to 11918+/- 1052 pixels per field for control mice; p = 0.003). Again there

was no significant difference in the vascular density in areas of normal brain adjacent to

the injury site (6621+/-289 pixels per field compared to 5191+/-1448 pixels per field;

p=0.15) (n= 5 for mice treated with liposomal clodronate and n=3 for control mice) (C).

Figure 3: Reformation of the blood brain barrier is delayed in mice treated with

liposomal clodronate. At various time points following hypothermic brain injury animals

were injected with FITC-labeled albumin and the leakage of albumin in the injured

hemisphere was calculated. Animals were injected intravenously with FITC-labeled

albumin. One hour later animals were injected intravenously with rhodamine-labeled

dextran (average MW approximately 70,000) and immediately sacrificed. The brains

were bisected and homogenized and the amount of FITC-albumin present in the

circulation was calculated using a ratio of rhodamine to fluorescene in the non-injured

hemisphere. The amount of FITC-albumin that had leaked into the parenchyma of the

injured hemisphere was then calculated in arbitrary fluorescence units. (A) In normal

controls a peak of albumin leak was observed on day two following injury with the

integrity of the BBB returning to baseline levels by day seven. (B) At day three

following injury the leakage of FITC-albumin was the same in both the liposomal

clodronate treated and control animals (301 +/- 126 compared to 259 +/- 153; p=0.7). A

non-significant trend toward increased FITC-albumin leak was seen in the liposomal

clodronate treated animals at day 5. At seven days after injury there was continued

leakage of FITC-albumin in the clodronate treated animals and minimal leakage in the

control animals (314 +/- 51 compared to 22 +/- 70; p=0.005). (n=4 mice in each group)

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