**4. SFKs regulate cell cycle for mitogenic toxicity and cell proliferation after ICH**

The cell cycle is an irreversible, ordered set of events that normally leads to cellular division [32-36]. The release of cells from a quiescent state (G0) results in their entry into the first gap phase (G1), during which the cells prepare for DNA replication in the synthetic phase (S). This is followed by the second gap phase (G2) and mitosis phase (M). After the cell has split into its two daughter cells, the new cells enter either G1 or G0. Mature neurons normally maintain themselves in G0 resting phase; however, a mature neuron that re-enters the cell cycle can neither advance to a new G0 quiescent state nor revert to its earlier G0 state. This presents a critical dilemma to the neuron from which death may be an unavoidable, but necessary, outcome for adult neurons attempting to complete the cell cycle [32,37]. Increasing evidence have revealed that aberrant cell cycle re-entry leads to neuronal death [8,32,37-64], and cell cycle inhibition via blocking SFKs can protect neurons from death post-ICH [8].

Apart from post-mitotic neurons, SFKs play critical roles in the process of cell cycle in dividable cells, by regulating mitogen-activated protein kinases (MAPKs) and cell cycle proteins such as cyclin-dependent kinases (Cdks) [65-69]. Although mitogenic signaling is necessary to initiate the cell cycle for normal cell division and proliferation, massive mitogenic signaling can also produce neurotoxicity and cell death [9,32,37,70]. Cell death and cell proliferation seem contradictory to each other, but these two seemingly different cellular processes share some common mitogenic molecules and signaling pathways (Fig. 1). In addition, many other molecules, including Ca2+, ROS, NO and MMPs can directly or indirectly activate or increase mitogenic signaling [54,71-77].

There are two stages (acute and recovery) after ICH (Fig. 1). Once ICH occurs, a large number of molecules (e.g., thrombin, glutamate, TNF-α, VEGF, etc.) are increased. This peaks within the first hour to a day in the acute stage after ICH, and then resolves gradually in the recovery stage after ICH. The over-activated SFKs/mitogenic signaling leads to neurons to enter the cell cycle and die, and damages astroctyes and BMVECs via MAPKs in acute stage after ICH. Within a day, however, the massive thrombin/SFK mitogenic signaling resolve, and the disease progresses to a recovery stage of ICH. The restored moderate SFK/mitogenic signaling leads to newborn BMVECs, astrocytes and other cells that mediate self-repair in the recovery stage after ICH.

As shown in Fig. 1, thrombin (a potent mitogen) triggers mitosis after ICH by modulating mitogenic intracellular molecules such as SFKs. SFKs participate in mitogenic signaling activation via regulating mitogen-activated protein kinases (MAPKs) and other molecules [64-69] that play critical roles not only in brain injuries during the acute stage in ICH, but in brain self-repair during the recovery stage in ICH. Acute inhibition of SFKs is beneficial, that attenuates hematoma, BBB breakdown, vasogenic edema, MAPK activation in the acute stage (0-24h) after ICH (Fig. 2, 3, 4, 5 & 6) [7-9,26,64,70]. In contrast, delayed and lasting inhibition of SFKs is detrimental, and prolongs BBB repair and brain edema resolution in the recovery stage (7-14 days) after ICH [9], presumably because SFKs mediate population of NPCs that exist in the "neurovascular niche". that repair the damaged BBB [78]. Such NPCs could serve as a source of newborn cells (i.e., BMVECs, astrocytes and perhaps other cells) of the neuro‐ vascular unit that play a role in re-establishing the BBB via the mitogenic growth signaling pathways during recovery phase after ICH (Fig. 1) [79].

**4. SFKs regulate cell cycle for mitogenic toxicity and cell proliferation after**

**Figure 2.** The effects of graded doses of SFK inhibitor PP2 (0.3 and 1.0 mg/kg, i.p.) and NMDA receptor inhibitor MK801 (1.0 mg/kg, i.p.) on injury produced by thrombin injections into striatum of Sprague-Dawley rats. **A.** Represen‐ tative Hematoxylin-Eosin stained section shows that 20U of thrombin causes brain injury, including hematoma and edema. **B.** Control injections of BSA into striatum produced minor injury. **C.** Infarction volumes 24h following striatum injections of thrombin compared to control BSA injections (BSA/Control) (n=6). Several groups of animals received striatal injections of thrombin: just thrombin alone (Throm/Saline) (n=6); prior intraperitoneal injection of PP2 (Throm/PP2/0.3mg/kg) (n=6); prior intraperitoneal injection of a higher dose of PP2 (Throm/PP2/1.0mg/kg) (n=6); and prior intraperitoneal injection of MK801 (Throm/MK801/1.0mg/kg) (n=6).. \* p<0.05 and \*\* P<0.01 vs Throm/ Saline (one-way ANOVA followed by Dunnett's *post hoc* test). **D.** PP2 (0.3 and 1.0 mg/kg, i.p.) and MK801 (1.0 mg/kg, i.p.) decrease the thrombin-induced motor deficits (n=9 for each group) using Elevated Body Swing Test (EBST) 23.5 hours after thrombin injections. Biased swinging behavior was calculated as follows: L/ (L+R) (%) for left biased swings (L, left-biased swings; R, right-biased swings). \* p<0.05 and \*\* P<0.01 vs Throm/Saline (one-way ANOVA followed by

The cell cycle is an irreversible, ordered set of events that normally leads to cellular division [32-36]. The release of cells from a quiescent state (G0) results in their entry into the first gap phase (G1), during which the cells prepare for DNA replication in the synthetic phase (S). This

**ICH**

Dunnett's *post hoc* test).

76 Intracerebral Hemorrhage

**Figure 4.** Intracerebroventricular injection of thrombin (20 U/animal, i.c.v.) causes reductions in astrocyte glial fibrillary acidic protein (GFAP) immunoreactivity after 1 day, and subsequent astrocyte proliferation around the brain vessels in lacunosum moleculare layer (LMol) of the hippocampus after 7 days and 14 days in Sprague-Dawley rats. **Panels A-C** show rats with sham operation labeled for BrdU (A), GFAP (B), and Merged image (C). GFAP+cells envelop most all of the brain vessel (arrows in panel B). **Panels D-F** show BrdU (D), GFAP (E) and the Merged image (F) at 1 day after thrombin injection. Compared with the sham group, there is decreased GFAP immunoreactivity around brain vessels.

(G), GFAP (H) and the Merged image (I) 1 day after thrombin plus PP2 injections. PP2 administration at day 0, immedi‐ ately after thrombin injection, blocks the thrombin-induced reductions in GFAP immunoreactivity. **Panels J-L** show the staining for BrdU (J), GFAP (K) and the Merged image (L) 7 days after thrombin injection. Compared to 1 day, BrdU +cells are increased 7 days after thrombin injection (J, arrows). Many of these BrdU+cells are co-labeled with GFAP (ar‐ rows in panel L). **Panel M** shows a higher power image of Panel L (area within dashed lines). GFAP stained astrocytes are red. The BrdU+GFAP+double-labeled new born astrocytic nuclei are yellow (arrows, Panel M). **Panels N-P** show the staining for BrdU (N), GFAP (O) and the Merged image (P) 14 days after thrombin injection. Compared to 7 days, BrdU +cells are decreased 14 days after thrombin injection. Some BrdU+cells (N, arrow) remain co-labeled with GFAP (arrow

cells located close to the vessel (arrows in panel F). **Panels G-I** show the staining for BrdU

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79

There are a few BrdU+/GFAP-

in panel P) 14 days after the thrombin injection. Scale bars: A-P, 50 µm.

**Figure 3.** Intracerebroventricular (i.c.v.) injection of thrombin (20 U/animal, i.c.v.) causes reductions in brain microvas‐ cular endothelial cell (BMVEC) immunoreactivity after 1 day, and subsequent BMVEC proliferation around the brain vessels in lacunosum moleculare layer (LMol) of the hippocampus after 7 days and 14 days in Sprague-Dawley rats. **Panels A-C** show rats following sham operations labeled for BrdU, bromodeoxyuridine (A), RECA-1, rat endothelial antigen-1 (B) and the overlay or Merged image (C). RECA-1+cells demonstrate the tube-shape of brain capillaries (ar‐ rows in panel C). **Panels D to F** show BrdU (D), RECA-1 (E) and the Merged image (F) at 1 day after thrombin injec‐ tions. Compared with the sham group, RECA-1+cells tend to lose their tube-shape at 1 day following thrombin injections (arrows in panel F) and there were no BrdU+cells co-labeled with RECA-1 at one day (panel F). **Panels G-I** show the staining for BrdU (G), RECA-1 (H) and the Merged image (I) 1 day after thrombin plus PP2 injections. PP2 administration at day 0, immediately after thrombin injection, blocks the thrombin-induced loss of tube-shape of RE‐ CA-1+cells. **Panels J-L** show the staining of BrdU (J), RECA-1 (K) and the Merged image (L) 7 days after thrombin injec‐ tion. Compared to 1 day, BrdU+cells are increased 7 days after thrombin injection. Some of these BrdU+cells are colabeled with RECA-1 (arrows in panel L and M). A few brain capillaries regained their tube-shape, though not completely. **Panel M** shows a higher power image of Panel L (area within dashed lines). RECA-1 stained BMVEC are red. The BrdU+/RECA-1+double-labeled new born BMVEC nuclei are yellow. **Panels N to P** show the staining for BrdU (M), RECA-1 (N) and the Merged image (P) 14 days after thrombin injection. Compared to 7 days, BrdU+cells are de‐ creased, but some BrdU+cells (N) remain co-labeled with RECA-1 (arrow in panel P), and more and more brain capilla‐ ries regained the tube-shape 14 days after the thrombin injection. Scale bars: A-P, 50 µm.

**Figure 4.** Intracerebroventricular injection of thrombin (20 U/animal, i.c.v.) causes reductions in astrocyte glial fibrillary acidic protein (GFAP) immunoreactivity after 1 day, and subsequent astrocyte proliferation around the brain vessels in lacunosum moleculare layer (LMol) of the hippocampus after 7 days and 14 days in Sprague-Dawley rats. **Panels A-C** show rats with sham operation labeled for BrdU (A), GFAP (B), and Merged image (C). GFAP+cells envelop most all of the brain vessel (arrows in panel B). **Panels D-F** show BrdU (D), GFAP (E) and the Merged image (F) at 1 day after thrombin injection. Compared with the sham group, there is decreased GFAP immunoreactivity around brain vessels. There are a few BrdU+/GFAPcells located close to the vessel (arrows in panel F). **Panels G-I** show the staining for BrdU (G), GFAP (H) and the Merged image (I) 1 day after thrombin plus PP2 injections. PP2 administration at day 0, immedi‐ ately after thrombin injection, blocks the thrombin-induced reductions in GFAP immunoreactivity. **Panels J-L** show the staining for BrdU (J), GFAP (K) and the Merged image (L) 7 days after thrombin injection. Compared to 1 day, BrdU +cells are increased 7 days after thrombin injection (J, arrows). Many of these BrdU+cells are co-labeled with GFAP (ar‐ rows in panel L). **Panel M** shows a higher power image of Panel L (area within dashed lines). GFAP stained astrocytes are red. The BrdU+GFAP+double-labeled new born astrocytic nuclei are yellow (arrows, Panel M). **Panels N-P** show the staining for BrdU (N), GFAP (O) and the Merged image (P) 14 days after thrombin injection. Compared to 7 days, BrdU +cells are decreased 14 days after thrombin injection. Some BrdU+cells (N, arrow) remain co-labeled with GFAP (arrow in panel P) 14 days after the thrombin injection. Scale bars: A-P, 50 µm.

**Figure 3.** Intracerebroventricular (i.c.v.) injection of thrombin (20 U/animal, i.c.v.) causes reductions in brain microvas‐ cular endothelial cell (BMVEC) immunoreactivity after 1 day, and subsequent BMVEC proliferation around the brain vessels in lacunosum moleculare layer (LMol) of the hippocampus after 7 days and 14 days in Sprague-Dawley rats. **Panels A-C** show rats following sham operations labeled for BrdU, bromodeoxyuridine (A), RECA-1, rat endothelial antigen-1 (B) and the overlay or Merged image (C). RECA-1+cells demonstrate the tube-shape of brain capillaries (ar‐ rows in panel C). **Panels D to F** show BrdU (D), RECA-1 (E) and the Merged image (F) at 1 day after thrombin injec‐ tions. Compared with the sham group, RECA-1+cells tend to lose their tube-shape at 1 day following thrombin injections (arrows in panel F) and there were no BrdU+cells co-labeled with RECA-1 at one day (panel F). **Panels G-I** show the staining for BrdU (G), RECA-1 (H) and the Merged image (I) 1 day after thrombin plus PP2 injections. PP2 administration at day 0, immediately after thrombin injection, blocks the thrombin-induced loss of tube-shape of RE‐ CA-1+cells. **Panels J-L** show the staining of BrdU (J), RECA-1 (K) and the Merged image (L) 7 days after thrombin injec‐ tion. Compared to 1 day, BrdU+cells are increased 7 days after thrombin injection. Some of these BrdU+cells are colabeled with RECA-1 (arrows in panel L and M). A few brain capillaries regained their tube-shape, though not completely. **Panel M** shows a higher power image of Panel L (area within dashed lines). RECA-1 stained BMVEC are red. The BrdU+/RECA-1+double-labeled new born BMVEC nuclei are yellow. **Panels N to P** show the staining for BrdU (M), RECA-1 (N) and the Merged image (P) 14 days after thrombin injection. Compared to 7 days, BrdU+cells are de‐ creased, but some BrdU+cells (N) remain co-labeled with RECA-1 (arrow in panel P), and more and more brain capilla‐

ries regained the tube-shape 14 days after the thrombin injection. Scale bars: A-P, 50 µm.

78 Intracerebral Hemorrhage

**5. Future directions**

been used in humans [82-85].

**Acknowledgements**

**Author details**

DaZhi Liu\*

**References**

Future studies need to address which specific SFK members found in brain (e.g., Src, Fyn, Lck and Yrk) that mediate ICH-induced cell death or birth. Since delayed and chronic inhibition of SFKs may impair neurogenesis and prolong BBB self-repair during recovery stage post-ICH, the acute and transient inhibition of SFKs should be pursued in treatment of ICH. The nanoparticle-based siRNA transfection system allows transient knockdown of target gene(s) and highly efficient delivery of siRNA *in vivo* with low cytotoxicity [80,81]. This could present a novel therapy for treating ICH patients as the nanoparticle-based siRNA approach provides heightened specificity for specific SFK gene(s) with less off target effects and this approach has

Src Family Kinases in Intracerebral Hemorrhage

http://dx.doi.org/10.5772/58488

81

The authors acknowledge the support of AHA Beginning Grant-in-Aid 12BGIA12060381 (DZL) and NIH grant NS054652 (FRS). Figures 2, and 3-6 were published in *Neurobiol Dis.*

Department of Neurology and the M.I.N.D. Institute, University of California at Davis Medical

[1] Sudlow CL, Warlow CP (1997) Comparable studies of the incidence of stroke and its pathological types: results from an international collaboration. International Stroke

[2] Flaherty ML, Haverbusch M, Sekar P, Kissela B, Kleindorfer D, et al. (2006) Long-

[3] Fogelholm R, Murros K, Rissanen A, Avikainen S (2005) Long term survival after pri‐ mary intracerebral haemorrhage: a retrospective population based study. J Neurol

term mortality after intracerebral hemorrhage. Neurology 66: 1182-1186.

*2008,30(2):201-11* and *Ann Neurol.* 2010,67(4):526-33, respectively.

Address all correspondence to: dzliu@ucdavis.edu

Incidence Collaboration. Stroke 28: 491-499.

Neurosurg Psychiatry 76: 1534-1538.

Center, Sacramento, California, USA

**Figure 5.** Brain sodium fluorescein (NF, **panel A**) and Evans blue (EB, **panel B**) extravasation increased 1 day after thrombin (Throm) injections (20U, i.c.v.), and decreased at 7 and 14 days in Sprague-Dawley rats. The thrombin inhibi‐ tor hirudin (Hir, 20U) blocked thrombin-induced NF/EB extravasation at 1 day after co-injection into the cerebral ven‐ tricles. PP2 (src family kinase inhibitor) administered with thrombin (day 0) blocked the NF/EB extravasation at 1 day after thrombin injection, whereas delayed PP2 administration (days 2-6) postponed alleviation of NF/EB extravasation at 7 days post-thrombin injection. Each column and vertical bar represents the mean ± standard error of the mean. \*\* p<0.01 vs. Cont; #p<0.05, ##p<0.01 vs. Throm/1day, ‡‡ p<0.01 vs. Throm/7days (one-way ANOVA followed by Tu‐ key's *post hoc* test).

**Figure 6.** Brain edema (water content) increased at 1 day after thrombin (Throm) injections (20U, i.c.v.), and decreased by 7 and 14 days in Sprague-Dawley rats. The thrombin inhibitor hirudin (Hir, 20U, i.c.v.) blocked elevation thrombininduced brain water content at 1 day after co-injection into the cerebral ventricle. Administration of PP2 (src family kinase inhibitor) at day 0 blocked the increase in brain water content observed at 1 day after thrombin injection, whereas delayed PP2 administration (days 2-6) prevented the resolution of brain water content at 7 days post-throm‐ bin injection. Each column and vertical bar represents the mean ± standard error of the mean. \*\* p<0.01 vs. Cont; #p<0.05, ##p<0.01 vs. Throm/1day, ‡ p<0.05 vs. Throm/7days (one-way ANOVA followed by Tukey's *post hoc* test).

## **5. Future directions**

Future studies need to address which specific SFK members found in brain (e.g., Src, Fyn, Lck and Yrk) that mediate ICH-induced cell death or birth. Since delayed and chronic inhibition of SFKs may impair neurogenesis and prolong BBB self-repair during recovery stage post-ICH, the acute and transient inhibition of SFKs should be pursued in treatment of ICH. The nanoparticle-based siRNA transfection system allows transient knockdown of target gene(s) and highly efficient delivery of siRNA *in vivo* with low cytotoxicity [80,81]. This could present a novel therapy for treating ICH patients as the nanoparticle-based siRNA approach provides heightened specificity for specific SFK gene(s) with less off target effects and this approach has been used in humans [82-85].

## **Acknowledgements**

The authors acknowledge the support of AHA Beginning Grant-in-Aid 12BGIA12060381 (DZL) and NIH grant NS054652 (FRS). Figures 2, and 3-6 were published in *Neurobiol Dis. 2008,30(2):201-11* and *Ann Neurol.* 2010,67(4):526-33, respectively.

## **Author details**

DaZhi Liu\*

**Figure 5.** Brain sodium fluorescein (NF, **panel A**) and Evans blue (EB, **panel B**) extravasation increased 1 day after thrombin (Throm) injections (20U, i.c.v.), and decreased at 7 and 14 days in Sprague-Dawley rats. The thrombin inhibi‐ tor hirudin (Hir, 20U) blocked thrombin-induced NF/EB extravasation at 1 day after co-injection into the cerebral ven‐ tricles. PP2 (src family kinase inhibitor) administered with thrombin (day 0) blocked the NF/EB extravasation at 1 day after thrombin injection, whereas delayed PP2 administration (days 2-6) postponed alleviation of NF/EB extravasation at 7 days post-thrombin injection. Each column and vertical bar represents the mean ± standard error of the mean. \*\* p<0.01 vs. Cont; #p<0.05, ##p<0.01 vs. Throm/1day, ‡‡ p<0.01 vs. Throm/7days (one-way ANOVA followed by Tu‐

**Figure 6.** Brain edema (water content) increased at 1 day after thrombin (Throm) injections (20U, i.c.v.), and decreased by 7 and 14 days in Sprague-Dawley rats. The thrombin inhibitor hirudin (Hir, 20U, i.c.v.) blocked elevation thrombininduced brain water content at 1 day after co-injection into the cerebral ventricle. Administration of PP2 (src family kinase inhibitor) at day 0 blocked the increase in brain water content observed at 1 day after thrombin injection, whereas delayed PP2 administration (days 2-6) prevented the resolution of brain water content at 7 days post-throm‐ bin injection. Each column and vertical bar represents the mean ± standard error of the mean. \*\* p<0.01 vs. Cont; #p<0.05, ##p<0.01 vs. Throm/1day, ‡ p<0.05 vs. Throm/7days (one-way ANOVA followed by Tukey's *post hoc* test).

key's *post hoc* test).

80 Intracerebral Hemorrhage

Address all correspondence to: dzliu@ucdavis.edu

Department of Neurology and the M.I.N.D. Institute, University of California at Davis Medical Center, Sacramento, California, USA

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**Chapter 7**

**Recovery from ICH – Potential Targets**

Intracerebral hemorrhage (ICH) is a devastating clinical event caused by rupture of blood vessels and accumulation of blood in the brain. Many disorders, including hypertensive arteriosclerosis, amyloid angiopathy, neoplasia, coagulation disorders and cerebrovascular malformations, directly or indirectly damage blood vessels in the brain and thus lead to ICH. The annual occurrence of ICH is estimated to be approximately 0.12 million in the USA and 2 million in the world. These numbers are expected to increase due to the aging of populations. Although accounting for only 15-20% of all strokes, ICH has severe clinical symptoms and poor prognosis. The 1-year survival rate of ICH is estimated to be 38% and long-term physical and mental disability is found in more than 90% of the survivors. Sadly, there is no effective treatment for ICH. Currently, primary supportive care and risk factor control are the main therapy for ICH in clinics. Thus, research and development of effective reagents to treat ICH is extremely urgent. In this chapter, we first introduce the anatomy and biology of the blood brain barrier. Then the pathophysiology and animal models of ICH are reviewed. Furthermore,

One unique feature about the blood vessels in the brain is the presence of the blood-brain barrier (BBB). BBB is a natural barrier that separates the central nervous system (CNS) from the circulation [1]. Under physiological conditions, the BBB prevents the entrance of blood cells and large molecules into the brain, but allows the uptake of nutrients and hormones from the blood, maintaining the homeostasis of CNS microenvironment [1, 2]. Under pathological conditions, the integrity of BBB is compromised and blood components leak into the brain, contributing to the progress of diseases [3-12]. At the cellular level, the BBB consists of brain

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

we summarize the potential therapeutic targets for ICH.

Yao Yao and Stella E. Tsirka

http://dx.doi.org/10.5772/58477

**1. Introduction**

**2. Blood brain barrier**
