Plexiform Lesions in an Experimental Model of Monocrotalin-Induced Pulmonary Arterial Hypertension

Abstract

Background: The monocrotaline (MCT)-induced pulmonary arterial hypertension model is one of the most reproduced today, presenting as a limitation the absence of plexiform lesions, typical manifestations of the severe disease in humans.

Objective: To evaluate the severity of MCT-induced pulmonary arteriopathy by pathological findings of lung and heart tissue samples, clinical course and 37-day survival.

Methods: Fifty male Wistar rats were divided into one of the four groups - control (CG) (n = 10) and three intervention (MCT) groups. The MCT groups received intraperitoneal injection (60 mg/kg) of MCT and remained exposed to the substance for 15 days (G15, n = 10), 30 days (G30, n = 10) and 37 days (G37, n = 20). At the end of each period, the animals were sacrificed, and pulmonary and cardiac tissues were collected for anatomopathological and morphometric analysis. The Kruskal-Wallis test was used, considering a level of significance of 5%.

Results: In the lungs of MCT animals, lesions related to pulmonary arteriopathy were found, including muscularization of the arterioles, hypertrophy of the middle layer and concentric neointimal lesions. Complex lesions were observed in MCT groups, described as plexiform and plexiform-like lesions. Right ventricular hypertrophy was evidenced by increased thickness and diameter of the cardiomyocytes and a significant increase in the right ventricular wall thickness (p <0.0000).

Conclusion: The MCT model was able to generate moderate-severe pulmonary arteriopathy associated with secondary right ventricular hypertrophy. The 37-day survival rate was 50%. To our knowledge, this study was the first to note the presence of complex vascular lesions, similar to those observed in patients with severe pulmonary arterial hypertension, in an isolated MCT model. (Arq Bras Cardiol. 2020; 115(3):480-490).

Figura 1. – Variação do peso corpóreo e peso relativo dos órgãos. Comparação entre os animais dos grupos controle (GC), 15 dias (G15), 30 dias (G30) e 37 dias (G37), ao final do período experimental. Em A, variação de peso corpóreo, em gramas, *p ≤ 0,0001; em B, peso relativo cardíaco (PCR), em g/100g de peso final (PF), *p < 0,005 e **p ≤ 0,0000; em C e D, peso relativo pulmonar esquerdo e direito (PPER e PPDR), em g/100g de peso final, *p ≤ 0,0000

Figura 2. – Fotomicrografias de tecido pulmonar do grupo controle. Observam-se alvéolos íntegros ocupando a maior parte do parênquima pulmonar (B e C) e arteríolas com estruturas e dimensões normais (A). Coloração hematoxilina-eosina; objetivas de 20X (A), 5X (B) e 2X (C)

Figura 3. – Achados histopatológicos vasculares e parenquimatosos do tecido pulmonar. Fotomicrografias de cortes histológicos do tecido pulmonar dos animais do grupo controle (GC) e de animais que receberam monocrotalina (grupos MCT); A – F, avaliação da remodelação arterial pulmonar observadas nas arteríolas alveolares vistas em cortes transversais (coloração de Weigert; objetiva 20X); G – I, alterações parenquimatosas nos animais dos grupos MCT (coloração hematoxilina-eosina; objetiva 1X, 5X, 5X, respectivamente); A e B, arteríolas alveolares do GC com estruturas e dimensões normais; C – F, arteríolas dos grupos 15, 30 e 37 dias; C e D, muscularização e hipertrofia da camada média, com proliferação neointimal concêntrica (D); E e F, lesões neointimais laminares celulares concêntricas com intensa redução do lúmen vascular e ruptura da membrana elástica externa (E); G, infiltrado inflamatório no interstício hilar, peribronquiolar e perivascular. Parênquima pulmonar apresenta poucas áreas alveolares aeradas associada a uma intensa exsudação intra-alveolar do tipo serofibrinosa (H) e macrofágica (I), com a presença de células esponjosas

Figura 4. – Lesões vasculares complexas do tecido pulmonar. Fotomicrografias representativas de lesões complexas do tecido pulmonar dos animais que receberam monocrotalina (grupos MCT) (coloração hematoxilina-eosina; objetiva 10X); A, arteríola alveolar do grupo controle com estrutura e dimensões normais; B – H, lesões “plexiform-like” (“stalk-like lesions”) em cortes transversais; B – D, arteríolas apresentando canais parecidos com fendas (“slit-like channels”), indicados pelas setas. E – H, arteríolas com pequenas massas de células hipercromáticas semelhantes a um broto (“bud-like”), projetando-se em direção ao lúmen vascular, indicadas pelas setas; I – M, lesões plexiformes em cortes transversais (grupo do dia 37, G37) mostrando a combinação de múltiplos pequenos canais capilares e pequenas fendas

Figura 5. – Evolução dos Parâmetros de Remodelação Vascular e Alterações Parenquimatosas. Avaliação Semiquantitativa (0 – sem alteração; 1 – discreto; 2 – moderado/acentuado)

Figura 6. – Achados histopatológicos do tecido cardíaco. Fotomicrografias de corte transversal (à esquerda) e longitudinal (à direita) do ventrículo direito de animais dos grupos controle (GC) e grupo 37 dias (G37) (coloração hematoxilina-eosina; objetiva 20X)

Figura 7. – Evolução da hipertrofia e dilatação do ventrículo direito. Fotomicrografias de corte transversal ao nível do 1/3 médio dos ventrículos cardíacos de animais do grupo controle (GC), 15 dias (G15), 30 dias (G30) e 37 dias (G37) (coloração hematoxilina-eosina)

Figura 8. – Comparação da espessura da parede do ventrículo direito e da área da câmara ventricular direita entre os grupos. Comparação da espessura da parede do ventrículo direito e da área da câmara ventricular direita entre os animais do grupo controle (GC), grupo 15 dias (G15), grupo 30 dias (G30) e grupo 37 dias (G37); *p < 0,0000

Figure 1. – Variation of body weight and relative weight of organs. Comparison between animals in the control group (CG), animals on day 15 (G15), on day 30 (G30) and on day 37 (G37). A, body weight variation, in grams, *p≤0.0001; B, relative cardiac weight (rHW), in g/100g of final weight (FW), *p<0.005 and **p≤0.0000; C - D, relative left and right lung weight (rLLW and rRLW), in g/100g of FW, *p≤0.0000.

Figure 2. – Photomicrographs of lung tissue in control group. Photomicrographs showing intact alveoli in most areas of pulmonary parenchyma (B and C) and arterioles with normal structures and dimensions (A). Hematoxylin-eosin staining; lens 20X (A), 5X (B) and 2X (C) objective lenses.

Figure 3. – Vascular and parenchymal histopathological findings of lung tissue. Photomicrographs of histological sections of lung tissue of animals of the control group (CG) and animals that received monocrotaline (MCT); A – F: cross sections showing pulmonary arterial remodeling in alveolar arterioles (Weigert’s stain; 20X objective lens); G – I: parenchymal changes in animals of the MCT groups (hematoxylin-eosin staining; 1X, 5X and 5X objective lenses, respectively); A and B alveolar arterioles with normal structure and dimensions; C – F, arterioles of groups 15, 30 and 37 days; C and D, muscularization and hypertrophy of the middle layer, with concentric neointimal proliferation; E and F, concentric, laminar neointimal lesions with important reduction of the vascular lumen and rupture of the external elastic membrane (E); G, peribronchial, perivascular, and hilar interstitial infiltrates; pulmonary parenchyma small areas of aerated alveoli associated with intense serofibrinous (H) and macrophagic (I) intra-alveolar exudation and sponge cells.

Figure 4. – Complex vascular lesions in lung tissue. Photomicrographs showing complex lesions in lung tissue samples of animals that received monocrotaline (MCT) (hematoxylin-eosin staining; lens 10X objective lens); A, alveolar arteriole in the control group with normal structure and dimensions; B – H, plexiform-like (“stalk-like”) lesions in cross sections; B – D, arterioles exhibiting “slit-like” channels (arrows); E – H, arterioles with “bud-like”, small mass of hyperchromatic and oval cells protruding from the arterial wall into the lumen (arrows); I – M, cross sections of plexiform lesions (animals on day 37, G37) showing a combination of multiple small capillary channels and narrow slits.

Figure 5. – Progression of vascular remodeling parameters and parenchymal changes. Semiquantitative assessment (0 – no change; 1 – slight; 2 – moderate/severe).

Figure 6. – Histopathological findings in cardiac tissue. Photomicrographs of cross (left) and longitudinal (right) sections of the right ventricle of animals in the control group (CG) and animals on day 37 (G37) (hematoxylin-eosin staining; lens 20X objective lens).

Figure 7. – Progression of right ventricular hypertrophy and dilation. Photomicrographs of cross sections at the mid-third of the ventricles of animals in the control group (CG), animals on day 15 (G15), day 30 (G30) and day 37 (G37) (hematoxylin-eosin staining)

Figure 8. – Comparison of right ventricular wall thickness and right ventricular chamber area between the groups. Comparison of right ventricular wall thickness and right ventricular chamber area between the animals of the control group (CG), animals on day 15 (G15), on day 30 (G30) and on day 37 (G37), *p < 0.0000.