Results

In situ expression of HNK-1 and p75^NTR in the adult canine nasal mucosa and olfactory bulb

HNK-1 and p75^NTR expression was studied in olfactory nerves (Fig.4-1a-e; Fig.4-2a-d,e-h, asterisk) and trigeminal nerve branches (Fig.4-2a-d, i-l) of the nasal mucosa as well as in the olfactory bulb (Fig.4-1f-i) and compared to OMP and MBP expression.

Anti-HNK-1- (Fig.4-1a) but not anti-p75^NTR-antibodies (Fig.4-1c) labeled olfactory nerve fascicles just beneath the olfactory epithelium (Fig.4-1a-e). Comparison of OMP immunoreactivity (Fig.4-1b,e), known to be associated specifically with olfactory neuron axons, together with distribution of HNK-1 in the olfactory epithelium (Fig.4-1d) demonstrated that HNK-1 was associated with olfactory neuron axons and not with OECs. Anti-HNK-1-antibodies identified a subset of olfactory neurons localized predominantly to the basal third of the epithelium (Fig.4-1d), while mature olfactory neurons defined by OMP expression occupied the middle third of the epithelium (Fig.4-1e). HNK-1⁺ neurons displayed a clear apical dendrite and a pronounced terminal knob (Fig.4-1d). P75^NTR immunoreactivity neither was associated with olfactory neurons nor with OECs but was found in connective tissue surrounding the olfactory nerves (Fig.4-1c).

To study the distal projection of the olfactory nerves in comparison with trigeminal nerve branches, the nasal septum close to the cribriform plate was stained for both antigens (Fig.4-2). While olfactory neuron axons containend OMP (Fig.4-2d,h), they were devoid of HNK-1 (Fig.4-2a,e), MBP (Fig.4-2b,f), and p75^NTR (Fig.4-2c,g) immunoreactivity. Contrary to this, myelinated trigeminal afferents (Fig.4-2i-l) identified by morhophological criteria and MBP immunoreactivity (Fig.4-2b,j) were positive for HNK-1 (Fig.4-2a,i) and p75^NTR 2c,k) and negative for OMP (Fig.4-2d,l). HNK-1 and p75^NTR in the trigeminal nerve brancheswere associated with myelinating (Fig.4-2i) and non-myelinating Schwann cells (Fig.4-2k), respectively. As observed in the lamina propria of the olfactory mucosa (Fig.4-1c), p75^NTR immunoreactivity was observed in connective tissue surrounding the nerves rather than in the olfactory nerves themselves (Fig.4-2c,g).

Olfactory neuron axons in the olfactory nerve layer (ONL) displayed OMP immunostaining (Fig.4-1g) but lacked HNK-1 (Fig.4-1f), which was found in the glomerular layer (GL) (Fig.4-1f) and external plexiform layer (EPL) as well as in deeper layers (not shown). A similar staining pattern was found for MBP which was absent from superficial layers but present in the mitral cell layer (MCL) and the EPL as well as in some processes reaching the GL (Fig.4-1h). No p75^NTR immunoreactivity was detected in the ONL or in deeper layers (Fig.4-1i).

In situ expression of HNK-1 and p75^NTR in the adult canine sciatic nerve and sympathetic trunk

To study the distribution of HNK-1 in mixed somatic and in autonomic nerves, the sciatic nerve (Fig.4-3a-f) and the cervicothoracic ganglion (Fig.4-3g-l) were analyzed for HNK-1 and p75^NTR immunoreactivity. HNK-1 expression in the sciatic nerve was specifically associated with myelinated nerve fibers (Fig.4-3a,d) identified by morphological criteria and MBP immunoreactivity (Fig.4-2b,e). No myelin sheaths were detected that lacked HNK-1 immunoreactivity (Fig.4-1a) implying that the entire population of myelinating Schwann cells expressed this carbohydrate epitope. Non-myelin-forming Schwann cells were visualized by anti-p75^NTR-antibodies (Fig.4-1c,f) and did not contain HNK-1.

In the cervicothoracic ganglion, as in the sciatic nerve, the distribution of HNK-1 completely overlapped with MBP (Fig.4-3g,j; Fig.4-3h,k) but not with p75^NTR (Fig.4-3i,l) suggesting that HNK-1/MBP and p75^NTR were specifically associated with preganglionic myelinated and postganglionic unmyelinated nerve fibers, respectively.

In addition to non-myelinated postganglionic nerve fibers, p75^NTR was found in the neuronal somata of the multipolar sympathetic neurons (Fig.4-3i).

Figure 4-1: Immunostaining of the adult canine olfactory mucosa (a-e) and olfactory bulb (f-i) with anti-HNK-1- (a,d,f), anti-OMP- (b,e,g), anti-p75^NTR- (c,i), and anti-MBP- antibodies (h).

HNK-1 and OMP but not p75^NTR immunoreactivity was associated with olfactory nerves close to the olfactory epithelium (a-c). In the olfactory epithelium, anti-HNK-1-antibodies reacted with a subset of olfactory neurons predominantly localized to the basal third of the olfactory epithelium (d) while anti-OMP-antibodies visualized mature olfactory neurons of the middle third of the epithelium (e). HNK-1⁺ neurons possessed a clear apical dendrite with a pronounced terminal knob (d). In the olfactory bulb, OMP was associated with olfactory neurons of the olfactory nerve layer (g), while HNK-1 (f) and MBP (h) were confined to deeper layers. P75^NTR was absent from all layers of the adult canine olfactory bulb (i). Scale bar in c,e = 25µm (a-e), scale bar in i = 100µm (f-i).

Figure 4-2: Immunostaining of trigeminal nerve branches (a-d, i-l), and olfactory nerves (a-d, asterisk, e-h) of the adult canine nasal septum with anti-HNK-1- (a,e,i), anti-MBP- (b,f,j), anti-p75^NTR- (c,g,k) and anti-OMP-antibodies (d,h,l). Olfactory nerves (asterisk) identified by OMP immunoreactivity (d,h) did not contain HNK-1 (e), MBP (f), and p75^NTR (g). P75^NTR immunoreactivity was detected in connective tissue surrounding the olfactory (c,g) and trigeminal nerve branches (c) as well as in non-myelinating Schwann cells of the trigeminal afferents (k) that were identified on the basis of MBP expression (b,j). Myelinating trigeminal Schwann cells were clearly positive for HNK-1 (a,i) but not for OMP (d,l). Scale bar in d = 100µm (a-d), scale bar in h,l= 25µm (e-l).

Figure 4-3: Immunostaining of the adult canine sciatic nerve (a-f) and cervicothoracic ganglion (g-l) with antibodies against HNK-1 (a,d,g,j), MBP (b,e,h,k), and p75^NTR (c,f,i,l). In the sciatic nerve, HNK-1 was present in all myelinated fibers (a,d) identified by anti-MBP-antibodies (b,e) whereas p75^NTR immunoreactivity was confined to the non-myelin-forming Schwann cell phenotype (c,f). The complete overlap in HNK-1 (g,j) and MBP (h,k) immunoreactivity in the cervicothoracic ganglion indicated that anti-HNK-1-antibodies identified myelinated preganglionic nerve fibers (g,j). Compared to this, p75^NTR displayed a reciprocal distribution (i,l). HNK-1^- and MBP^- nerve fibers together with neuronal somata of the ganglion stained positive for p75^NTR suggesting that the neurotrophin receptor was specifically associated with non-myelinated postganglionic nerve fibers (i,I). Scale bar in c,i=

100µm (a-c, g-i), scale bar in f,l= 20µm (d-f, j-l).

In vitro expression of HNK-1 and p75^NTR in trigeminal Schwann cells and olfactory bulb-derived olfactory ensheathing cells

In vitro expression of HNK-1 and p75^NTR in OECs and Schwann cells was both studied in primary culture (Fig.4-4-4-6) and in anti-p75^NTR-immunopurified preparations following passaging (Fig.4-7). To separately analyze the expression of HNK-1 and p75^NTR in vitro, OECs and Schwann cells were isolated from the adult canine olfactory bulb (Krudewig et al. 2006) and maxillary nerve, respectively, and not from the nasal mucosa which is known to harbor both cell types. The suspensions were seeded onto 96-well microtiter plates and double-immunostained for HNK-1/p75^NTR and HNK-1/MBP at day 3 and 7 in vitro either as live cells or after fixation and permeabilization. Parallel to this, OECs and Schwann cells were immunopurified from the mixed cultures using magnet-activated cell sorting (Wewetzer et al., 2005; Krudewig et al., 2006) and expanded in the presence of HRG-1ß. Double-immunostaining for HNK-1/p75^NTR was done 1 week after the first passage.

Spindle-shaped cells clearly identified as Schwann cells based on their morphological phenotype after 3 and 7 days in vitro (Fig. 4-4a-d; Fig.4-6a,b) and after the first passage (Fig.4-7a-d) consistently expressed p75^NTR. The staining was confined to the entire cell surface of fixed (Fig.4-4a-d; Fig.4-6a,b, Fig.4-7a,b) and viable cells (Fig.4-7c,d). No differences in the staining pattern were found between total cell cultures (Fig.4-4a-d; Fig.4-6a,b) and immunopurified preparations (Fig.4-7a-d). Contrary to this was the distribution of HNK-1, which at both 3 and 7 days in vitro was found associated with perinuclear vesicles of p75^NTR-positive and negative cells (Fig.4-4, arrows) rather than with the entire cell. In addition to this, HNK-1 was found associated with cellular fragments either adhering to the cell surface (Fig.4-4a-d, arrowheads in a, c) or lacking cellular contact. Moreover, only fixed and permeabilized cells but not live Schwann cells (Fig.4-6a-d) stained positive for HNK-1. HNK-1 immunoreactivity was lost from cultures from about day 8-10 on. Neither fixed (Fig.4-7a,b) nor live p75^NTR-expressing Schwann cells (Fig.4-7c,d) after the first passage stained positive for HNK-1.

MBP expression in maxillary nerve cultures 4e-h) was similar to HNK-1 (Fig.4-4). MBP immunoreactivity was either associated with cellular fragments (Fig.4-4e,f, arrowheads) or associated with perinuclear vesicles of fixed (Fig.4-4e-h, arrows) but not with those of viable p75^NTR-expressing Schwann cells (Fig.4-6c,d).

Doublestaining for HNK-1 and MBP clearly demonstrated that the majority HNK-1⁺ vesicles was MBP^- (Fig.4-4e-h) indicating that both antigens were not present within the same vesicles. Neither HNK-1 (Fig.4-6a-d) nor MBP (Fig.4-6c,d) was detectable on live Schwann cells implying that both antigens displayed an intracellular distribution.

Expression of HNK-1, p75^NTR and MBP in OECs from the olfactory bulb followed the same spatiotemporal pattern as described above for Schwann cells. Expression of p75^NTR was detected in all cells of a spindle-shaped phenotype at day 3 and 7 (Fig.4-5a-d; Fig.4-6e,f) and maintained after purification and passaging (Fig.4-7e-h).

Staining for p75^NTR was not dependent on permeabilization of the cells and found both in fixed and viable cell cultures (Fig.4-5a-d; Fig.4-6e,f; Fig.4-7e-h). HNK-1 was found in association with cellular fragments adhering to the cell surface and perinuclear vesicles at day 3 (Fig.4-5a,b,e,f, arrows) or in association with MBP positive fragments (Fig.4-5e,f, arrowheads) and was hardly detectable at day 7 (Fig.4-5c,d,g,h). No HNK-1 immunoreactivity was detectable in purified OECs after the first passage (Fig.4-7e-h). As in Schwann cells, MBP was found in p75^NTR- expressing OECs in association with vesicles. At day 7, MBP was not detectable (Fig.4-5g,h; Fig.4-6g,h).

Figure 4-4: Double-immunostaining of fixed adult canine maxillary nerve Schwann cells in mixed culture with antibodies against HNK-1/p75^NTR (a-d) and HNK-1/MBP (e-h) three (a,b,e,f) and seven days (c,d,g,h) after seeding. Schwann cells with the typical spindle-shaped morphology displayed p75^NTR expression along the entire cell body (a,c) whereas HNK-1⁺ material was found in small spots at the soma (arrows in a,c,e) with a presumptive intracellular distribution as well as in cellular fragments adhering to the external cell surface (arrowheads in a-d). MBP immunoreactvitiy was associated with HNK-1⁺ myelin fragments (arrowheads in e,f) as well as with small spots at the cell soma (arrows in e,g). Scale bar in h = 60µm (a-h).

Figure 4-5: Double-immunostaining of fixed adult olfactory bulb-derived olfactory ensheathing cells in mixed culture with antibodies against HNK-1/p75^NTR (a-d) and HNK-1/MBP (e-h) three (a,b,e,f) and seven days (c,d,g,h) after seeding. OECs with a spindle-shaped morphology after three and seven days displayed p75^NTR expression along the entire cell body (a,c) whereas HNK-1⁺ material after seven days was found in small spots at the soma with a presumptive intracellular distribution (arrow in c). After three days in vitro, HNK-1-immunoreactivity was found in small spots at the soma (arrow in e) as well as associated with cellular debris adhering to the external cell surface (arrow in a, arrowheads in e,f).

MBP immunoreactivity was associated with HNK-1⁺ myelin fragments (arrowheads in e,f) as well as with small spots at the cell soma (arrow in e). MBP⁺ material was not detectable at seven days in vitro (g). Scale bar in h = 60µm (a-h).

Figure 4-6: Double immunostaining of live adult canine maxillary nerve Schwann cells (a-d) and olfactory bulb-derived olfactory ensheathing cells (e-h) in mixed culture with antibodies against HNK-1/p75^NTR (a,b,e,f) and HNK-1/MBP (c,d,g,h) seven days after seeding. Both Schwann cells and OECs at seven days in vitro were stained with anti-p75^NTR-antibodies (a,e) but not with anti-HNK-1- (a-h) and anti-MBP- antibodies (c,g) implying that p75^NTR and HNK-1/MBP displayed an extracellular and intracellular distribution, respectively. This conclusion was based on the comparison between live and permeabilized cell staining of Fig.5. Scale bar in h = 60µm (a-h).

Figure 4-7: Double-immunostaining of purified fixed (a,b,e,f) and live (c,d,g,h) adult canine maxillary nerve Schwann cells (a-d) and olfactory bulb-derived OECs (e-h) after passaging with antibodies against HNK-1/p75^NTR (a-h). Both fixed and live Schwann cells (a-d) and OECs (e-h) expressed p75^NTR (a,c,e,g) but not HNK-1 (a,c,e,g). Scale bar in h = 60µm (a-h).