For this edition of the Journal Club, Dr Marvin Firth, BVSc (Hons), DipRCPath, DipFMS, AFHEA, MRSB, MRCVS, breaks down research conducted by Ushio et al. (2021). This paper was brought to us by the pathologists working at the University of Tokyo – the aim of their research was to characterise arteriolar lesions in the testes, epididymides and ovaries of dogs and evaluate the relationship between the presence of these arteriolar lesions and other pathological changes.
Background – why was this study conducted?
Apart from in birds, rabbits and pigs, arteriolar pathology is not commonly seen in domestic animals. Instead, it is in humans that we have observed atherosclerosis in relation to ischaemic heart disease and infarctions of the myocardium and cerebrum.
The deposition of lipids and infiltration of amorphous eosinophilic deposits (composed of fibrin, glycosaminoglycans, immunoglobulins and, less commonly, small quantities of amyloid) leads to hyalinosis. There are questions surrounding whether hyalinosis could be a precursor to atherosclerosis. Interestingly, arteriosclerosis of the gonads has been reported in buffalos, pigs, horses, primates and humans.
In this paper, Ushio et al. note that gonads removed in a variety of dogs often had similar changes in their arteriolar walls. As this discovery had never been reported in dogs, the authors set about compiling the following data and images.
Materials and methods
One hundred and thirty-nine testes and epididymides from 72 males (0.42 to 14.1 years old) and 200 ovaries from 105 females (0.58 to 16.0 years old) were retrieved from the sample archive at the University of Tokyo.
As well as routine haematoxylin and eosin staining, arteriolar lesions were subjected to special staining protocols, including Elastica van Gieson, Congo red and oil red O stains. Immunohistochemistry (IHC) was also performed to determine the distribution of lipid-laden foamy cells and apolipoproteins.
A statistical analysis was then performed to assess any relational effects, including those related to age.
Pathology detected in the test series included:
- Evaluation of 139 testes and epididymides from 72 dogs:
- seminiferous tubule atrophy and degeneration (n=68)
- seminoma (n=20)
- interstitial tumour (n=20)
- Sertoli cell tumour (n=8)
- orchitis (n=1)
- necrosis (n=1)
- Evaluation of 200 ovaries from 105 female dogs:
- persistent corpus luteum (n=130)
- rete ovarii cysts (n=41)
- cysts of subsurface epithelial structures (n=40)
- extra ovarian cyst (n=33)
- Graafian follicular cyst (n=13)
- corpus luteum cyst (n=10)
- granulosa cell tumour (n=7)
- epithelial cell tumour (n=4)
The paper produced some beautiful histological images depicting the full range of gonad arteriolar changes, lesions, special stains and IHC.
The testes and epididymides
The arteriolar lesions in the testes and epididymides were classified into four types according to their histopathological features. These included fibromuscular hypertrophy, vasculitis, vacuolar changes and hyalinosis (Figure 1;1 to 6); these categories were also used for the ovarian tissues examined.
Fibromuscular hypertrophy was characterised by thickening of the tunica intima with collagen fibre and smooth muscle (Figure 1;1 and 2).
Vasculitis was recognised by the infiltration of mononuclear cells (Figure 1; 3 and inset). In severe cases of vasculitis, the arteriolar layers were obscured (Figure 1; 4) and shown using the Elastica van Gieson stain.
Vacuolar change was characterised by thickening of the tunica intima with the infiltration of oil red O-positive lipid-laden foamy cells and the deposition of oil red O-positive granules (Figure 1; 5a and 5b). In some lesions with vacuolar change, the internal elastic lamina was disrupted or duplicated, and the tunica media was severely thin (Figure 1; Elastica van Gieson-stained 5c).
Hyalinosis was characterised by the irregular thickening of the tunica intima with Congo red-positive amyloid deposits (Figure 1; 6a and the polarised section in 6b). Oil red O-positive granules were colocalised with amyloid deposits (Figure 1; 6c), and the internal elastic lamina was disrupted or duplicated in the hyalinosis lesions (Figure 1; 6d).
Overall, fibromuscular hypertrophy, hyalinosis and vacuolar change were observed in the epididymis and testis, but vasculitis was only observed in the epididymis.
Figure 2 shows the histological images of the sections taken from the ovaries, looking at the same features – fibromuscular hypertrophy, vasculitis, vacuolar changes and hyalinosis (Figure 2;7 to 10).
Fibromuscular hypertrophy was seen, with the irregular thickening of the tunica intima (Figure 2; 7).
Vasculitis was also appreciated (Figure 2; 8a), with the mononuclear infiltrate clear in the inset image. Arteriolar layers were, again, obscured in the Elastica van Gieson stain (Figure 2; 8b).
Vacuolar change was noted via the deposit of oil red O-positive granules (Figure 2; 9a and 9b), with further internal elastic lamina disruption and duplication also observed (Figure 2; 9c).
Hyalinosis was seen with Congo red-positive amyloid deposits (Figure 2; 10a and 10b), and, again, the colocalisation of oil red O-positive granules with amyloid (Figure 2; 10c) was observed. Finally, hyalinosis was also seen to disrupt the internal lamina (Figure 2; 10d).
Immunohistochemical analysis of arteriolar changes
Looking at the immunohistochemical analysis of vacuolar changes and hyalinosis, the cells infiltrating the tunica intima were immunopositive for alpha-SMA (smooth muscle actin) and Iba-1 (Figure 3; 11 to 14). Some of those cells were swollen and vacuolated.
The authors state that the number of Iba-1-positive foamy cells in vacuolar changes of the testes was larger than that of alpha-SMA-positive foamy cells (Figure 3; 11). However, this author is not sure if the images provided show the opposite! Meanwhile, the number of alpha-SMA-positive foamy cells in vacuolar changes of the ovaries was larger than that of Iba-1-positive foamy cells (Figure 3;13), which is what this author believes the image shows.
Immunoreactivity for apoA1, apoE, apoB, oxPL and LOX1 was observed in the amyloid deposits and subendothelial spaces of the tunica intima (Figure 4; 15 to 18). Strong cytoplasmic immunoreactivity for apoA1, apoE, apoB, oxPL and LOX1 was observed in the infiltrated foamy cells of the tunica intima in ovaries, while foamy cells in the testes were positive for apoE and negative for all other markers. The staining intensity for apoE and apoB was greater in amyloid deposits than apoA1.
Conclusion and take-home messages
Overall, this was an interesting paper which stemmed from the observations of diagnostic pathologists.
The authors concluded that early changes in the arteriolar structures of gonads should lead to further research as they are possibly indications of underlying vascular health in a variety of species, but especially in the dog, where such changes had not been previously reported.
My take-home messages from this paper include:
- Fibromuscular hypertrophy, vasculitis, vacuolar change and hyalinosis are histopathological features of arteriolar lesions that can be appreciated in canine gonads
- Hyalinosis and vacuolar change in the testes/epididymides and ovaries are the depositions of lipids and proteins related to atherosclerosis
- Ageing is likely an important risk factor for arteriolar lesions with lipid deposits of the canine gonads
- Fibromuscular hypertrophy is more common in ovaries than testes/epididymides
- Extra-ovarian cysts are significantly related to the presence of arteriolar lesions without lipid deposits
- Vasculitis is more common in epididymides than ovaries
- Foamy smooth muscle cells are part of vacuolar change, with immunohistochemistry revealing apoA1, apoE, apoB, oxPL and LOX1 in ovaries, but apoE only in testes. Similarly to humans, vacuolar change in foamy smooth muscle cells may, therefore, be a precursor of atherosclerotic plaques
- Dogs with interstitial cell tumours in the testes have lower serum testosterone than normal dogs
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