DATOS ESPECÍFICOS DE LA PROPUESTA 2.1 Justificación:

NO has been implicated in a number of neurodegenerative diseases including ALS. There is evidence that NOS is upregulated in motor neurons in adult SOD1mutant mice at early presymptomatic (<24 weeks) and during symptomatic (>32 weeks) stages of the disease (Sasaki et al., 2002). However, little is known about the expression of NOS in the postnatal SOD1mutant, thus, a preliminary investigation was made to assess NADPH diaphorase reactivity in the SOD1 G93A mouse model of ALS.

NADPH diaphorase reactivity was recorded in the grey matter of the lumbar spinal cord of both SOD1G93A mice and wild type littermates at P12. The pattern of reactivity in both SOD1 WT and SOD1 G93A mice was consistent with that observed in CD-1WT mice. No difference in the total number of NADPH diaphorase reactive neurons was noted between SOD1 WT and CD-1WT mice (two-way ANOVA with Bonferroni multiple comparison post-hoc test). However, the total number of NADPH diaphorase reactive neurons was significantly lower in the P12 SOD1G93A when compared to the SOD1 WT mice (Student’s t-test, P<0.05).

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Dorsal Horn (Lamina I – VI)

The laminae of the dorsal horn contain the largest population of NADPH diaphorase reactive neurons (Table 2.6). Reactive neurons in both SOD1 WT and SOD1G93A sections did not appear to differ in size from CD-1WT neurons. NADPH diaphorase reactive neurons constitute a similar percentage of the total population of reactive neurons in the dorsal horn of both SOD1 WT and SOD1G93A mice (approximately 60% of the total segmental population; Table 2.6). However, there were less reactive neurons overall in the rostral segments L1 and L2 of SOD1G93A, when compared to SOD1 WT mice. The total number of reactive neurons in SOD1 WT mice was 68±1 in L1 and 79±2 in L2 compared to 53±1 in L1 and 49±1 in L2 of SOD1G93A mice (Table 2.5).

The number of reactive neurons increased in the cord along the rostral-caudal axis with the largest population recorded in the caudal levels of both SOD1 WT and SOD1G93A mice (Fig. 2.12A; Table 2.5.).

Lamina VII

NADPH diaphorase reactive neurons were again distributed throughout lamina VII with a trend towards the largest population being located within the first three rostral segments of the spinal cord. NADPH diaphorase reactive neurons comprised a similar percentage of the total population of reactive neurons in lamina VII of both SOD1 WT and SOD1G93A mice, except in L2 where a higher proportion of the total reactive population were found in lamina VII of SOD1G93A mice compared to SOD1 WT mice (approximately 25% of the total segmental population in the SOD1G93A and approximately 17% in the wild type; Table 2.6). However, there were less reactive neurons in L1 and L2 of the SOD1G93A, when compared to the SOD1 WT. The total number of reactive neurons in the SOD1G93A mouse was 23±1 in L1 compared to 30±1 in L1 of the wild type littermates (Table 2.5; Fig. 2.13C).

The sympathetic preganglionic neurons were again observed as clusters in the IML of upper lumbar segments (L1-L2 only) (Fig. 2.11A). Interestingly, the level of NADPH diaphorase reactivity in the IML was approximately 16-fold lower in SOD1G93A than in

53 SOD1 WT, particularly at L2 (P<0.05, Mann-Whitney test, SOD1G93A, n=4 and WT, n=2; Fig. 2.12A and B, Fig. 2.12D; Table 2.5).

Lamina VIII and IX

Very few neurons were NADPH diaphorase reactive in laminae VIII and IX (less than one per segment). The lamina VIII neurons were found mostly in the rostral segments of the SOD1 WT. In lamina X, only one reactive neuron was recorded in the SOD1 WT while none were recorded in the SODG93A (Tables 2.5). Light punctate staining in the areas around the approximate location of the motor pools was noted occasionally in both SOD1 WT and SODG93A sections.

Lamina X

The population of NADPH diaphorase reactive neurons in lamina X remained relatively constant along the rostral-caudal axis and no difference was noted between the SOD1 WT and SOD1G93A groups (Table 2.5 and 2.6). The neurons of the lumbar dorsal commissural nucleus were recorded in the cell count for lamina X as described in the CD-1WT. NADPH diaphorase reactive neurons constitute a similar percentage of the total population of reactive neurons in lamina X of both SOD1 WT and SOD1G93A, with the exception of L5. In L5 the SOD1G93A had a lower proportion of the total reactive population compared to the SOD1 WT (approximately 16% of the total segmental population in the SOD1G93A and approximately 21% in the wild type; Table 2.6). The total number of reactive neurons in the SOD1G93A mouse was 17±1 in L5 compared to 23±1 in L5 of the wild type littermates (Table 2.5; Fig. 2.13B). NADPH diaphorase reactive neurons comprised a similar percentage of the total population of reactive neurons in L2 of both SOD1 WT and SOD1G93A but the total number of reactive neurons in the SOD1G93A mouse was lower in L2 compared to the wild type littermates (17±1 and 27±1, respectively; Table 2.5).

In both SOD1 WT and SOD1G93A, the pattern of NADPH diaphorase reactive neurons closely resembled that of the CD1 WT mouse. There were overall less NADPH diaphorase reactive neurons in the SOD1G93A compared to the SOD1 WT mouse, in all the laminae described and particularly in the rostral regions (L1 and L2).

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Figure 2.2. Transverse section taken at P3L1 demonstrates NADPH diaphorase reactivity in lumbar spinal cord. A. Discrete populations of neurons stain throughout the spinal cord section, stained neurons are visible in the dorsal Ai, intermediolateral layer Aii and lamina X (LX) Aiii. High magnification (x40) brightfield images of typical neurons in the dorsal horn with dorsoventral projections B, Ai., sympathetic preganglionic neurons in theintermediolateral layer project laterally and are closely associated with the vasculature C, Aii and neurons in LX with both dorsoventral and lateral projections. Staining appears to be restricted to the cytoplasm in all lamina expect those of the superficial dorsal horn which appear to stain in both nucleus and cytoplasm. D, Aiii. Vascular staining is visible, throughout the section (black arrowheads).Annotations:cc – central canal,d – dorsal, DF – dorsal funiculus, LF – lateral funiculus, v – ventral,VF – ventral funiculus, VMF – ventral median fissure. Scale bars = 200µm, A; 50µm, B, C and D.

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Figure 2.3. Schematic diagram of NADPH diaphorase activity in the mouse spinal cord at P1-3 taken from spinal segments lumbar (L)1-5. Ai-Ei. Approximate positions of positively stained neurons are indicated by black filled circles. Each lumbar segment represents 5 transverse segments from 1 mouse and each filled circle represents one neuron. d and v indicate dorsal and ventral orientation. Diagrams are not to scale. Aii-Eii. Graphs showing the total number of positively stained neurons in LX, LVII, D and the total in LVII which include the SPN cells of the IML (TVII for L1-3 only). Data are mean ±SEM, n=6.

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Figure 2.4. Transverse section taken at P5L1 demonstrates NADPH diaphorase reactivity in lumbar spinal cord. A. Discrete populations of neurons stain throughout the spinal cord section, stained neurons are visible in the dorsal Ai, intermediolateral layer Aii and lamina X (LX) Aiii. High magnification (x40) brightfield images of typical neurons in the dorsal horn with dorsoventral projections B, Ai., sympathetic preganglionic neurons in theintermediolateral layer project laterally and are closely associated with the vasculature C, Aii and neurons in LX with both dorsoventral and lateral projections. Staining appears to be restricted to the cytoplasm in all lamina expect those of the superficial dorsal horn which appear to stain in both nucleus and cytoplasm. D, Aiii. Vascular staining is visible, throughout the section (black arrowheads).Annotations:cc – central canal,d – dorsal, DF – dorsal funiculus, LF – lateral funiculus, v – ventral,VF – ventral funiculus, VMF – ventral median fissure. Scale bars = 200µm, A; 50µm, B, C and D.

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Figure 2.5. Schematic diagram of NADPH diaphorase activity in the mouse spinal cord at P4-6 taken from spinal segments lumbar (L)1-5. Ai-Ei. Approximate positions of positively stained neurons are indicated by black filled circles. Each lumbar segment represents 5 transverse segments from 1 mouse and each filled circle represents one neuron. d and v indicate dorsal and ventral orientation. Diagrams are not to scale. Aii-Eii. Graphs showing the total number of positively stained neurons in LX, LVII, D and the total in LVII which include the SPN cells of the IML (TVII for L1-3 only). Data are mean ±SEM, n=6.

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Figure 2.6. Transverse section taken at P7L1 demonstrates NADPH diaphorase reactivity in lumbar spinal cord. A. Discrete populations of neurons stain throughout the spinal cord section, stained neurons are visible in the dorsal Ai, intermediolateral layer Aii lamina X (LX) Aiii and lamina VII Aiv. High magnification (x40) brightfield images of typical neurons in the dorsal horn with dorsoventral projections

B, Ai., sympathetic preganglionic neurons in the intermediolateral layer project laterally and are closely