Biochemical Assessments of Cerebral Vasospasm: Measurement of cGMP, PKC, and PTK in Cerebral Arteries - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 512

and connective tissue (e.g., collagen). Tunica adventitia, with the
exception of neurotransmitters released from sympathetic and
parasympathetic nerves, has been considered as a physical supporter
of arteries. However, recent reports suggest that cells located
within adventitia can also produce vasoactive substances and regu-
late arterial diameter ( 5 ). As a whole, smooth muscle contraction/
relaxation dictates arterial diameter while the endothelium and
adventitia regulate smooth muscle contraction/relaxation via the
release vasoactive substances.
In this chapter, we focus on several key intracellular signaling
molecules that have been implicated in the pathological constriction
of cerebral arteries (cerebral vasospasm) occurring after subarach-
noid hemorrhage (SAH); cyclic guanosine monophosphate (cGMP),
protein kinase C (PKC), and protein tyrosine kinase (PTK). We
provide detailed background information about these molecules
and describe techniques and typical procedures used to measure
the activities of these molecules in the cerebral vasculature of SAH
model animals.

The canine “two-hemorrhage” model ( 6 ) is used as an animal SAH
model. Animals undergo surgery for injection of autologous arte-
rial blood into the cisterna magna on days 1 and 4. Following
euthanasia of SAH model animals, craniotomy is performed and
the brainstem containing the basilar artery is excised from the
brain, as quickly as possible. The tissue is immersed in dissecting
buffer solution (in mM: 128.9 NaCl, 4.10 KCl, 1.54 NaH 2 PO 4 ,
1.01 CaCl 2 , 1.19 MgSO 4 , 24.9 HEPES, and 10 glucose; pH 7.4
with NaOH). With the aid of a dissecting microscope, the basilar
artery is removed from the brainstem. Blood is then fl ushed from
the lumen of the artery and the arachnoid membrane and extravas-
cular blood is meticulously removed. After denudation of the
endothelium, the artery is either used immediately, or fl ash frozen
in liquid nitrogen and stored at −80°C until assayed.

1.1. Tissue Collection

1.2. Protein Assay

The protein concentration of each sample is determined using
Protein Assay reagent (Bio-Rad), which is based on Bradford assay
( 7 ). Each sample is diluted (1:100) with distilled water to avoid
any potential impact of chemicals contained in extract buffer on
the protein assay. Bovine serum albumin is used as a standard (0,
50, 100, and 150 mg/mL). Samples and standards (40 mL each)
are placed in a 96-well microplate, and 160 mL of diluted Protein
Assay reagent is added to each well. After incubation at room tem-
perature for 5 min, the absorbance is measured at 595 nm by a
microplate spectrophotometer. The protein concentrations are cal-
culated from the standard curve, and used for normalization of
values obtained from assays.