**3. SFKs modulate NMDA receptor for brain injury after ICH**

mediated mitogenic growth; (3) Nerve growth factor (NGF), epidermal growth factor (EGF)

**Figure 1.** Src family kinases (SFKs) participate in mitogenic signaling pathways that play critical roles in blood-brain barrier (BBB) disruption and self-repair after intracerebral hemorrhage (ICH). The network does not apply to certain cell type(s). The arrows do not necessarily indicate direct binding and/or activation of the downstream molecules; in‐

Src family kinases (SFKs), a family of proto-oncogenic proteins, participate in both neurotoxicity at acute stage and neurogenesis during recovery stage post-ICH (Fig 1). Our previous studies have confirmed the time-specific and conflicting roles of SFKs using their inhibitor (PP2) after ICH: (1) Acute administration of PP2 (immediately post-ICH) decreases local cerebral glucose utilization (LCGU), activity of SFKs, attenuates BBB breakdown, brain edema, and cell death around ICH and improves behavioral function following ICH [6-9]; (2) Chronic inhibition of PP2 (2-6 days) blocks BBB repair and brain edema resolution in the recovery stage

SFKs are a family of non-receptor protein tyrosine kinases, include nine family members Src, Fyn, Lck, Lyn, Yes, Hck, Blk, Fgr, and Yrk [10-12], of which Src, Fyn, Yes and Yrk widely

**2. Tissue specificity, structure and activity regulation of SFKs**

termediate proteins or kinases may exist.

74 Intracerebral Hemorrhage

(7-14 days) after ICH [9].

and other growth factor-mediated neurogenesis; and others (Fig. 1).

NMDA receptors are ionotropic glutamate receptors, comprise NR1, NR2 and NR3 subunits, which form the central conductance pathway [22,23]. In the physiological conductions, activation of NMDA receptors results in the opening of an ion channel that allows the flow of Na+ and small amounts of Ca2+ into the cell and K+ out of the cell [22,23]. Following ICH there is a transient increase of glutamate release and local cerebral glucose utilization in the region surrounding the ICH, and the antagonists of NMDA receptors reverse the glucose hypermetabolism produced by ICH [6]. However, glutamate alone could not explain the hyperme‐ tabolism since glutamate injected directly into brain did not produce hypermetabolism [6]. Apart from glutamate release, ICH may affect NMDA receptors in some way to make them more sensitive to glutamate in order to mediate injury and/or hypermetabolism.

ICH activates SFKs [7,8], and SFK members (e.g., Src, Fyn) up-regulate the ion channel activity of NMDA receptor and make them more sensitive to glutamate by phosphorylating the NR2A and NR2B subunits of the NMDA receptors [19,24-27]. It has been proved that phosphorylation by Src at Tyr-1292, Tyr-1325 and Tyr-1387 in NR2A subunit increases activity of NMDA receptors, and phosphorylation of tyrosine residues by Src in the C-terminal of the subunits prevents a Zn2+-dependent inhibition of the NMDA receptors and thus increases channel conductivity [28-30]. We have demonstrated that either NMDA receptor inhibitors (MK-801) or SKF inhibitors (PP2) can attenuate brain injury at the acute stage after ICH (Fig. 2) [8]. These results suggest that either activation of NMDA receptors or SFKs is sufficient to produce brain injury post-ICH.

NMDA receptor activation has also been shown to enhance NPCs proliferation and lead to increased neurogenesis [31]. However, there is no direct report showing the mechanism by which SFKs participate in NMDA receptors mediated neurogenesis after brain injury.

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

Src Family Kinases in Intracerebral Hemorrhage

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

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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

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

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].

cycle inhibition via blocking SFKs can protect neurons from death post-ICH [8].

mitogenic signaling [54,71-77].

after ICH.

**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 Dunnett's *post hoc* test).
