6.2.4
βAPP immunoreactivity
Brain and spinal cord sections were examined under the light microscope. Line drawings of each brain section were used to record the type and amount of immunoreactivity, with each tissue section being divided into a number of neuroanatomical regions. The amount of immunoreactivity present in each region was rated on a semi-quantitative scale similar to that described by Gentleman et al. (1995) and Reichard et al. (2003a).
Details of this scoring system are summarised in Table 6.1.
0 no positive staining
+ (mild) low numbers of individual positively stained axons usually requiring a 40x objective for good visualisation
++ (moderate) scattered clusters of positively stained axons, or moderate numbers of individual positively stained axons
+++ (marked) extensive staining of large areas of white matter, visible using a 1.5x objective
Table 6.1 Key for semi-quantitative scoring of anti-bAPP immunoreactivity in brain sections.
Figure 6.1 (below) shows examples of moderate and marked anti-bAPP immunoreactivity.
Figure 6.1. Examples of immunoreactivity scored as ‘++’ (moderate; A) and ‘+++’ (marked; B). Both images were captured using a 10x objective for comparison. Anti-bAPP immunohistochem- istry. Bars = 100 mm.
Composite scores were assigned by adding the scores in each neuroanatomical region to give a total axonal injury score for each animal. Results for individual pups were translated to an electronic format using digitised line drawings created on Xara software. For each case, the distribution and amount of anti-bAPP immunoreactivity in each brain section was mapped using greyscale values as shown in Figure 6.2.
Figure 6.2. An example of mapping bAPP scores using greyscale values. Scores are indicated using in-
creasingly dark shades of grey, as shown on the key in the middle of the lower panel.
Slides were examined and mapped prior to review of the gross necropsy findings and histological brain lesions associated with the case. Patterns of immunoreactivity defined by Reichard et al. (2003a) and Blumbergs et al. (2008) were used as a basis of classification. This method describes five patterns:
(i) Diffuse traumatic axonal injury
A history of trauma, in conjunction with scattered individual swellings/bulbs or
20% black (+) 40% black (++) 90% black (+++)
groups of swellings/bulbs in the corpus callosum, cerebral hemispheric white matter and brainstem, in a pattern that is not consistent with vascular axonal injury (see below).
(ii) Multifocal traumatic axonal injury
A history of trauma, in conjunction with scattered/groups of swellings or bulbs in the corpus callosum and hemispheric white matter, but not in the brainstem, in a pattern that is not consistent with vascular axonal injury.
(iii) Vascular axonal injury
A ‘zig-zag’ like pattern of immunoreactivity, often seen as clearly demarcated areas within white matter tracts, caused by compromise of the vascular supply due predominantly to increased intracranial pressure.
(iv) Metabolic axonal injury
Scattered immunopositive axons in the absence of a history of trauma, and not in a pattern of vascular axonal injury, probably due to hypoglycaemia or, more rarely, global hypoxia-ischaemia (Dolinak et al. 2000a, 2000b; Reichard et al. 2005).
(v) Penumbral axonal injury
Axonal immunoreactivity surrounding a focal lesion.
This classification method could not be directly applied to the sea lion pups as no history was available, thus excluding diffuse axonal injury and multifocal traumatic axonal injury from classification. Furthermore, because it was not possible to definitively exclude trauma, metabolic axonal injury could also not be diagnosed. Instead, a new category of ‘multifocal’ axonal injury was used to describe cases with widespread white matter immunoreactivity that was not distributed in a pattern suggestive of vascular axonal injury. A number of pups also had a periventricular pattern of immunoreactivity, which was included as a separate category. Thus the modified classification system used in this study was as follows:
(i) Vascular axonal injury: clusters of immunopositive axons arranged in a zig-zag like pattern, as confirmed by D. Blumbergs, pers. comm.
(ii) Multifocal axonal injury: scattered clusters or individual immunopositive axons arranged in a pattern not consistent with vascular axonal injury.
(iii) Penumbral axonal injury: immunopositive axons arranged around the margins of a focal lesion.
(iv) Periventricular axonal injury: immunopositive axons surrounding a ventricle. Pups that had only a few scattered positive axons (scored as ‘+’) in a few sections were classified as having ‘minimal’ overall immunoreactivity. ‘Widespread’ axonal injury was defined as the presence of moderate or marked immunoreactivity in a non-vascular pattern, in the cerebellar peduncles and brainstem, and at two or more levels of the corpus callosum and cerebral white matter, with a total axonal injury score of 20 or more.
Anti-βAPP immunoreactivity of neurons was evaluated manually using a light microscope. Cerebellar sections (block 9) were scanned using a 40x objective lens, and three sets of 100 Purkinje cells were counted using non-overlapping fields. Immunopositive Purkinje cells were defined as those with anti-βAPP positive granules occupying more than 50% of the neuronal soma, and were expressed as an average percentage of the three counts.
Neurons in the frontal cortex and brainstem were assessed for immunoreactivity of the neuronal soma, using a semi-quantitative scale: ‘0’ (no immunoreactivity), ‘+’ (less than 25% of neurons immunopositive), ‘++’ (approximately half of neurons immunopositive) or ‘+++’ (over 75% of neurons immunopositive).
A 4mm grid graticule was placed over sections of cortex and brainstem to evaluate neuronal soma immunoreactivity as described by Finnie et al. (2010). Grid squares
were assessed as positive if they contained one or more neurons with immunopositive granules occupying more than 50% of the soma.
Eye sections were evaluated for the presence, amount and location of anti-βAPP immunoreactivity, as well as the type of axonal profile.