Generation of an INOS knockout mouse was first described by Wei and colleagues 97 The iNOS gene was disrupted in embryonic stem cells by homologous recombination with a replacement-targeting construct. The resultant clone was used to produce germ line chimaeras. Mice heterozygous for the mutation were then bred to obtain homozygotes for the disrupted iNOS gene (‘knockout’).
Homozygous animals are viable and fertile with no evidence of
histopathology within the major organs. However, with disruption of the iNOS gene, there is absence of stimulated macrophage release of NO which is cytostatic and cytotoxic to protozoan parasites, fungal cells and bacteria. Consequently, these animals are susceptible to diseases related to these organisms. For instance, the wild-type mice and the heterozygous mice are highly resistant to Leishmania major infection, and all animals achieved spontaneous healing after footpad innoculation. In contrast, the mutant mice are highly susceptible to the infection and developed visceral disease 97
NO from iNOS is known to be released in large quantities in the vasculature in endotoxaemia and the stubborn loss of vascular tone
contributes towards the high mortality in septicaemic shock. iNOS mutants have been shown to be resistant to liposaccharide-induced death compared to wild-types, which developed severe loss of mass when injected intra- peritoneally with liposaccharide 97
Altered immunity and capacity to mount an effective inflammatory response in these mutants are consistent with our present understanding of the role of iNOS in the reticulo-endothelial system. However, the impact of iNOS absence on the neoinitmal vascular response to localised injury is unknown and it was the aim of this part of the study to address this issue using iNOS mutants as an investigative tool.
4.3.2 Methods
Animals & operative procedure- iNOS mutants for the present study were contributed by Dr.D.Rees (Clinical Pharmacology, UGL). These mutants were derived by targeted iNOS gene disruption in C57BL/6J strain mice by Laubach and collègues ^8 and have been imported from Jackson Laboratory, Maine, USA (www.jax.org). The wildtype C57BL/6J mice were used as
experimental controls and are referred to here as ‘B110’s.
iNOS knockout male (n=12) mice weighing between 25 g to 33 g underwent left carotid injury using the tungsten wire method as previously described in Section 4.2.2. In order to exclude variations in injury response that might occur between animal strains, a control injury group (n=13) was also established using the background strain from which the iNOS knockouts were developed (B110).
Tissue harvest and processing- Two weeks post-injury, animals were killed then perfusion fixed by infusing 4% formal-saline, at 90 mmhg, through a 24-gauge butterfly needle in the left ventricle. Carotids from both sides were removed and immersed in the same fixative for a further 24 hours and
then processed for paraffin sectioning. 5 pm sections were taken every 100 pm throughout the length of tissue and were stained with H&E (Fig 22). In sections where injuries were identified by breaks in the elastic laminae, the adjacent sections were also stained with EVG.
iNOS immunohistochemistrv- Additional animals underwent tungsten wire injury (n=2 each for iNOS and B110 groups). At day 7 post-procedure, injured carotids were removed and immediately frozen in OCT. 5 micron cryostat sections were cut and fixed in 2% formaldehyde / 0.2%
glutaraldehyde for 20 minutes. Sections were washed in TBS (x3, 5 min each), then exposed to goat serum, 1:20 for 30 minutes in order to block non specific protein binding sites. Rabbit anti-mouse iNOS primary antibody (Transduction laboratories) was applied for 1 hour at room temperature, followed by alkaline phosphatase labelled goat anti-rabbit IgG (Dako).
Specific enzyme activity was revealed with Vector Red (Vector). Non-specific tissue alkaline phosphatase activity was inhibited by the addition of levimisole (Dako) to the chromogenic substrate. TBS rinses (x3) were instituted
between each antibody step. Finally, slides were counterstained with Mayer’s haematoxylin.
Morphometrv- A single lesion was defined as 'a 100 micron length of artery that contained section(s) with disrupted elastic lamina'. The following
Fig 22 - Scheme for carotid tissue processing and morphometry.
Upper panel- Diagram illustrates a longitudinal length of an injured common carotid artery. Paraffin sections were taken every 100 pm (shaded region) through the entire length, for H&E staining. In regions where sections contained disruptions of the elastic lamina, neointimal lesions were also found and parallel sections were stained with EVG for morphometric analysis. A single neointimal lesion was defined as a ‘100 pm’ length of vessel containing section(s) with neointima. Thus, in this example, if sections in region ‘A’ and ‘C’ contained neointimal thickenings, the carotid artery was marked as possessing two lesions.
Lower panel- Schematic cross-section of an injured carotid artery, containing a lesion. Neointimal lesions were always localised to the area of elastic lamina breach. The severity of injury was classified by the ‘depth of injury’ (ELI, EL2 or EL3) and also, by the ‘breadth of injury’ (expressed as a % of the lumenal circumference).
The following cross-sectional areas were measured: neointimal, lumenal, medial and adventitial. Neointimal measurements were expressed as neointimal / medial area ratios.
B D III ' ...:__ <- 100 microns 4- 100 microns 4- 100 microns
^^Adventitia ^ Elastic laminae
external elastic lamina
lumen
Neointima
injury score
1. Depth of injury. ^L1 (innermost elastic lamina breached)