Novel therapeutic potential of angiotensin receptor 1 blockade in a rat model of diabetes-associated depression parallels altered BDNF signalling

Figures

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  Fig. 1. Losartan attenuates depressive behaviour of diabetic rats. OFT and FST were performed on control (non-diabetic) rats, vehicle-treated diabetic rats (DM) and losartan-treated diabetic rats (DM+LOS). (a) In OFT, locomotor activity is represented by number of grid crossings. (b–f) In FST, floating reflects immobility (b), while time spent in active mobility (c) is represented by struggling (d), swimming (e) and diving (f). Each moving pattern is represented as percentage of time. Data are presented as mean ± SD (n = 6–8/group). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control rats; †p < 0.05 vs DM

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  Fig. 2. Decreased cerebral perfusion of diabetic rats is unaltered by losartan. (a) In control (non-diabetic) rats, vehicle-treated diabetic rats (DM) and losartan-treated diabetic rats (DM+LOS), mean arterial pressure was calculated. (b, c) Cerebral blood perfusion was measured by SPECT–MRI based on [99mTc]HMPAO uptake. Representative images from different sections of the whole brain. Colours represent the magnitude of [99mTc]HMPAO uptake (green, purple and yellow represent low, middle and high uptake, respectively) (b). Quantified data revealed significantly decreased perfusion in all investigated brain regions in DM and DM+LOS rats vs controls (c). (d, e) Cerebrovascular activation was assessed by ICAM-1 immunostaining in coronal brain sections of the dentate gyrus. Images are representative. Scale bar, 50 μm. (d) Integrated density of fluorescent images was determined by using Image J and expressed as arbitrary units (e). (f, g) Protein levels of p-eNOS (f) and endothelin-1 (g) in rat hippocampus were also evaluated (fold change vs control, which was set as 1). Ponceau S total protein staining was used as loading control (for a representative example of Ponceau S-stained membranes,…

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  Fig. 3. Losartan mitigates proinflammatory cytokine responses in diabetic rats. Neuroinflammation was assessed by various methods in control (non-diabetic), vehicle-treated diabetic rats (DM) and losartan-treated diabetic rats (DM+LOS). (a, b) Microglia activation was measured indirectly by SPECT–MRI based on [125I]CLINME uptake. (a) Representative images from different sections of the whole brain. Colours represent the magnitude of [125I]CLINME uptake (green, purple and yellow represent low, middle and high uptake, respectively) (b) Quantified data revealed significantly increased CLINME uptake in all investigated brain regions in DM and DM+LOS rats. (c) Microglial activation was assessed by P2Y12 immunostaining. Arrowheads showing P2Y12-positive microglia with thicker cell bodies and a partial withdrawal of processes (indicative of higher level of microglial activity). Scale bar, 50 μm. Quantification was based on unbiased densitometric analysis of P2Y12 signals in coronal brain sections of the dentate gyrus. (d) GFAP immunostaining showing astrocyte responses to diabetes and losartan and unbiased densitometric analysis showing reduced GFAP levels in coronal brain sections of the…

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  Fig. 4. BDNF production and localisation is normalised by losartan in diabetic rats. (a–f) Representative blot (a) and quantification of protein levels of proBDNF (b), mBDNF (c) and cleavage enzymes (d–f) in the hippocampus of control (non-diabetic) rats, vehicle-treated diabetic rats (DM) and losartan-treated diabetic rats (DM+LOS). Data are presented as fold change vs control, which was set as 1. Ponceau S total protein staining was used as loading control (for a representative example of Ponceau S-stained membranes, see ESM Fig. 3b). (g) Triple immunofluorescence staining showing BDNF, GFAP-positive astrocytes and Iba1-positive microglia in the brain. Arrowheads in inserts indicate astrocytes with BDNF immunopositivity. Representative images of coronal brain sections of the dentate gyrus from three rats (representing each group) are shown. Scale bar, 200 μm. Data are presented as mean ± SD (n = 6–8/group). *p < 0.05, **p < 0.01 and ***p < 0.001 vs control rats; †p < 0.05 and †††p < 0.001 vs DM

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  Fig. 5. Losartan alters neuronal responses via mBDNF–TrkB signalling. (a–f) Representative blot and protein levels of p75NTR (b), p-JNK (c), TrkB (d), p-ERK (e) and p-CREB (f), in the hippocampus of control, diabetic (DM) and losartan-treated diabetic (DM+LOS) rats. Ponceau S total protein staining was used as loading control (for a representative example of Ponceau S-stained membranes, see ESM Fig. 3b). (g, h) mRNA expression levels of Bax (g) and Bcl2 (h) in the hippocampus. mRNA expression of Bax and Bcl2 was normalised to Rn18s mRNA expression. In (a–h), Data are expressed as fold change vs control, which was set as 1. (i) Immunofluorescence images showing a marked upregulation of p-CREB in the hippocampal dentate gyrus in response to treatment with losartan. Integrated density of fluorescent images was determined using Image J software. Densitometric analysis showed a significant difference in p-CREB levels in DM+LOS compared with control and DM. No colocalisation of p-CREB with P2Y12-positive microglia (green) or blood vessels identified by tomato lectin (blue) can be seen. Scale bar, 100 μm. (j) p-CREB staining shows colocalisation with neurons identified by the pan-neuronal…