Fig 2: Immunohistochemical iNOS labelling observed in the brain slices obtained through sections S4 and 5.Section S4 (bregma -4.80 mm): absence of iNOS-positive cells in V2L area of control rats, contrary to infected rats who exhibit iNOS-positive neurons (black arrow and dots); magnification (X3) is shown; in control rats, hippocampus CA1 area is free of iNOS-positive cell labelling; in infected rats, CA1 pyramidal neurons exhibit iNOS-positive labelling (black arrow, also in X3 magnification). Hippocampal (CA1, CA2, CA3 and DG) and cortical (PtA and V2L) areas exhibit only iNOS positive neurons (black dots). This is also the case for LH and SNR structures. iNOS positive neuroglial cells and neurons are both present in PAG (red and black dots). Section S5 (bregma -3.14, ventral part): in control rats, Pe nucleus is free of iNOS-positive cellular elements, contrary to infected rats (Pe nucleus with labeled iNOS-positive astrocytes and microglial cells (red arrows, also in X3 magnifications); in the schema, Arc, PeF, VMH, SOR and Amyg exhibit mixed labelling (neurons, black dots, and neuroglial cells, red dots). Again, piriform (Pir) and ectorhinal (ECt) cortices exhibit only iNOS positive neurons (black dots). Abbreviations and signs see Fig 2 and reference [25].

Fig 3: Identification of brain cells (neurons, astrocytes and microglial cells) expressing the inducible nitric oxide synthase (iNOS) in Wistar male rats 16 days after infection with T. b. brucei.A–Time course of parasitemia after infection (mean value ± SEM, n = 6, per day); trypanosomes appear in the blood 6 days (D6) later, corresponding to stage 1 human African trypanosomiasis (HAT); successive parasitemic waves take place; abscissae: days after infection; ordinates: parasitemia/mL. B—Immunofluorescent staining of T. b. brucei (in red, white arrow) in the brain parenchyma on D16 post-infection, corresponding to stage 2 of HAT (section S5, subfornical organ of the hypothalamus, n = 3), bregma -3.14 mm (according to reference [25]); C—Hypothalamic cell nuclei stained with DAPI (blue, subfornical organ of the hypothalamus); T. b. brucei are also stained (white arrows); D—Sagittal schema of the brain (lateral 0.4 mm / (according to reference [25]) showing the position of the 5th coronal section (S5) chosen for iNOS immunostaining; E and F—Coronal schemas at bregma -3.14 mm (according to reference[25]) showing ventral (E) and dorsal (F) diencephalon. Grey matter, containing all brain cell types, was examined; G—Cellular labelling at section S5, bregma -3.14 using specific biological markers: neurons (NeuN), astrocytes (GFAP) and microglia (integrin alpha M) were identified (left column of part G); the 3 types of cells were also iNOS-positive (medial column of part G); double labelling appears in the right column (Neurons: NeuN and iNOS; Astrocytes: GFAP and iNOS; Microglia: Integrin alpha M and iNOS). Abbreviations: T. b. brucei, Trypanosoma brucei brucei; DAPI, 4’, 6’ Di amino-2-phenylindole; DNA, deoxyribonucleic acid; scale is given in µm, micrometer; NeuN, neuronal specific nuclear protein in vertebrates; GFAP, glial fibrillary acidic protein; Pe, periventricular hypothalamic n; PVP, paraventricular thalamic n, posterior part; SOR, supraoptic n, retrochiasmatic; An Olympus BX51 microscope was equipped with DP50 camera (objective 64x10).

Fig 4: Immunohistochemical iNOS labelling observed in the brain slices obtained through sections S1, S2 and S3.Section S1 (bregma -13.24 mm): absence of iNOS-positive cells in Cu area of control rats, contrary to infected rats (iNOS-positive astrocytes, red dots, and neurons, black dots); magnifications (X2) are shown; in infected rats, IOD, IRt, LRt, and SolM exhibit only iNOS-positive neurons (black dots; for histological data see File 1-Supporting Information). Section S2 (bregma -11.30 mm): in control rats (also in X3 magnification), cerebellar lobule areas 4&5 do not reveal iNOS-positive cells; in infected rats, this area shows a layer of iNOS-positive Purkinje neurons (black arrows, also in X3 magnification); infected rats exhibit iNOS-positive neurons in LVe, MVeMC and SpVe (black dots in schema). Section S3 (bregma -8.72 mm): in control rats, PnR is free of iNOS-positive cells, opposite to infected animals (iNOS-positive neurons, black dots, black arrows), who also exhibit DRC iNOS-positive neurons (black dots); infected animals, exhibit both iNOS-positive astrocytes (red dots) and neurons (black dots) in CnF, PAG, Tz, tz and ml. Abbreviations and signs: see Fig 2 and reference [25].

Fig 5: Brain distribution of iNOS-positive neurons (black dots), astrocytes and microglial cells (red dots).S1-8 –Sagittal schema of the brain (lateral 0.4 mm; according to [25]) showing the 8 sections (S1-S8) performed in 4 rats. The sections extended from bregma +1.20 mm to bregma -13.24 mm and were analyzed for the distributions of the cells. The iNOS-positive astrocytes and microglial cells are mainly located in periventricular structures and basal parts of the brain. The cerebral cortex remains devoid of iNOS-positive astrocytes and microglial cells (S4, S5, S6, S7 and S8). Abbreviations: n, nucleus; 4&5, Cerebellar lobules; AcbC, accumbens n, core; AHA, anterior hypothalamic area, anterior part; AID, agranular insular cortex, dorsal; AIP, agranular insular cortex, posterior; Amyg, amygdala; Arc, arcuate n; AUD, secondary auditory cortex, dorsal; CA1, CA2, CA3, hippocampus fields (corni ammonis CA1, CA2 and CA3); Cortex, cerebral cortex; Cereb, cerebellum; Cg1, cingulate cortex, area 1; Cg2, cingulate cortex, area 2; CnF, cuneiform n; CPu, caudate putamen; Cu, cuneate n; CxA, cortex amygdala transition zone; DG, dentate gyrus; DI, dysgranular insular cortex; DRC, dorsal raphe n, caudal part; Ect, ectorhinal cortex; GI, granular insular cortex; Hipp, hippocampus; Hypo, hypothalamus; IG, granular insular cortex; IOD, inferior olive, dorsal n; IRt, intermediate reticular n; LA, lateroanterior hypothalamic n; LGP, lateral globus pallidus; LH, lateral hypothalamic area; LRt, lateral reticular n; LSD, lateral septal n, dorsal part; LVe, lateral vestibular n; M1, primary motor cortex; M2, secondary motor cortex; Midb, midbrain; ml, medial lemniscus; ml, Medial Lemniscus; MO, medulla oblongata; MVeMC, medial vestibular n, magnocellular part; PAG, periaqueductal gray; Pe, periventricular hypothalamic n; PeF, perifornical n; Pir, piriform cortex; PnR, pontine raphe n; PRh, perirhinal cortex; PtA, parietal association cortex; PVA, paraventricular hypothalamic n, anterior part; PVP, paraventricular thalamic n, posterior part; RCh, retrochiasmatic area; RSGb, retrospenial granular b cortex; S1, primary somatosensory cortex; S1BF, primary somatosensory cortex, barrel field; S1FL, primary somatosensory cortex, forelimb region; S1HL, Primary somatosensory cortex, hindlimb region; S1J, primary somatosensory cortex, jaw region; S1JO, primary somatosensory cortex, jaw region; S1Tr, primary somatosensory cortex, trunk region; S1ULp, primary somatosensory cortex, upper lip region; S2, secondary somatosensory cortex; SCh, suprachiasmatic n; SHi, septohippocampal n; SNR, substantia nigra; SO, supraoptic n; SolM, solitary n medial tract; SOR, supraoptic n, retrochiasmatic; SpVe, spinal vestibular n; Thal, thalamus; Tz, trapezoid body n; V2L, secondary visual cortex, lateral area; VDB, n of vertical limb diagonal band; VMH, Ventromedial thalamic. For a more detailed description of the structures, see [25].