MARCO CONCEPTUAL

E13 & E14

At the earliest stage examined there is complete absence of GAP 43 in the spinal cord except for the developing Bundle of His, although staining is visible in the white matter. The Bundle of His is the consolidation of dorsal root fibres that have grown towards the spinal cord but not yet penetrated it, during this period before they enter the spinal grey matter they travel up and down the spinal cord. 24 hours later the Bundle of His is more intensely stained and extends medially with one or two axons moving down the lateral side of the cord. The dorsal roots are quite discernible but loosely bundled, as are the ventral roots. The most significant growth at E14 is the faint but intricate tangle of axons in the ventral horn. This is mainly restricted to the most lateral portion, with some medially adjacent to the developing ventral columns. Some faint fibres are observed crossing the ventral commissure.

PGP 9.5 results are similar to those with GAP 43, showing no axonal staining in the grey matter at E l 3. The ventral spinal cord does display some diffuse cellular stain which on the lateral edges extends more dorsally. By E l 4, there are few differences except that some ipsilateral projection neurons have extended a few short axons medially.

Peripherin at E l 3 shows much clearer development of ventral columns including fibres transversing the spinal cord. These appear to be ipsilateral projection neurons. The ventral grey matter is also more clearly defined with axonal labelling in the lateral portion only. At E14, the Bundle of His has developed medially leading with individual axons. Importantly, a single axon also descends ventrally from the most lateral and ventral part of the Bundle of His. Ventrally, the lateral staining has intensified and spread dorsally with the appearance of a few strongly staining cell bodies and faint fibres just ventral and lateral to the central canal. The main finding using this marker compared to GAP 43 or PGP 9.5, is the presence of cell bodies in the ventral horn at E l 4.

At E l 3, RT97 labelling is restricted to the Bundle of His, which is much smaller than observed with the above markers, and punctate staining where the lateral and ventral

columns will form. However, a large number of changes are seen within 24 hrs. By E14, The dorsal roots are closely bundled to meet the Bundle of His which appears in a more rostral position. A very significant finding is the appearance of RT97-IR fibres in the dorsal horn of the spinal cord grey matter. Fibres enter and travel ventrally remaining close to the lateral wall. This is before innervation is observed in this location with any marker other than the few peripherin positive fibres. RT97 is also a unique marker at this developmental stage due to the intensity of individual fibres, although no cell bodies are detected.

No labelling in the dorsal horn was obtained with SP nor with CGRP at E13. CGRP, a peptide normally found in adult small diameter DRG neurons, is also found in motoneurons and it is present in the lateral ventral horn at E14. This is the only region in which CGRP is detectable. At higher magnification, this labelling is distinguishable in cell bodies. Using trkA however, no labelling is found in the spinal cord. The dorsal roots are numerous and heavily labelled but loosely bundled to the extent that practically all fibres are individually discernible. These form the Bundle of His at the edge of the spinal cord which is positioned slightly more laterally into the connective tissue. By E14 little has changed, the cord is still absent of any innervation, change is limited to tighter fasciculation of the dorsal roots and Bundle of His.

E15 & E16

At E l 5, there is clear growth of dorsal root collaterals into the grey matter of the dorsal horn. GAP 43 labelling shows the Bundle of His has moved further medially to form the dorsal columns. The most lateral aspect of these give rise to afferents that project in a ventrolateral arc towards the lateral columns - these correspond to future Ap fibres. In the ventral horn there are some faintly labelled fibres that are situated at the most ventral aspect, and in addition, some fibres cross the ventral commissure. The ventral roots are still loosely fasciculated. By E l6 , the dorsal columns have approached the midline but do

not meet. The lateral column is well formed but stainiqg is less intense in the region of the dorso-lateral funiculus. At higher magnification of the dorsal horn, the afferents

and terminate near the dorso-lateral funiculus in the lateral grey matter. Fibres are also observed at the top of the central canal which extend ventrally either side of it. The ventral horn labelling is unchanged apart from a decrease in intensity at the ventral commissure.

PGP 9.5 labelling at E15 is mostly unchanged but there is some faint evidence of one or two axons projecting from the dorsal column into the grey matter ventro-laterally towards the lateral grey matter, corresponding to future la fibres. A similar picture is found at E l6 except that cellular staining increases in the dorsal horn.

Peripherin at E15 shows axonal labelling in the dorsal horn, but this displays a different pattern and is derived from a different location than seen with GAP 43. The afferents invade the dorsal horn from the medial part of the dorsal column. These arc medially, then ventrally into the mid-dorsal horn. Associated with these afferents is some cellular staining, mostly in the superficial dorsal hom. At the same time axons also invade the grey matter from the lateral walls of the spinal cord, these are short and very fine and follow a torturous path with some ending in bulbous swellings. These may be presumptive ipsilateral projection neurons. In the ventral hom, the pool of axonal staining is situated in a more medial position than at E14 but the cellular label is unchanged. By E l6 the dorsal horn labelling retains the same pattern but extends deeper into the dorsal

horn. The ventral motor pools are less intensely labelled.

RT97-IR afferents in the E15 dorsal horn have changed their expression pattern considerably since E14 and now more closely resemble the pattern of GAP 43. The ventro-laterally orientated afferents meet another numerous group of axons originating from the lateral cord that project towards the ventricular zone. The ventrally directed axons could be presumptive AB afferents, but they have not as yet turned laterally. The white matter is also labelled strongly and delineates the spinal cord, enclosing the highly complex tangle of axons in the ventral horn. This network of fibres is located mostly in the lateral and ventral region. Fibres are also identified horizontally crossing the ventral commissure. By E l6 , collaterals in the dorsal horn now originate from the medial dorsal

hom, some follow the pattern of the previous afferents but a few continue ventrally. The projections towards the ventricular zone have advanced medially and dorsally. The other

anomalous observation is some stray axons moving ventrally from the medial edge of each dorsal column. Meanwhile in the ventral horn, axons feed into the ventral commissure and cross to the contralateral side.

There is still no CGRP labelling in the dorsal horn while immunopositive cells appear more dorsally in the ventral hom. TrkA labelling is still absent from the grey matter at E l6 . The dorsal columns are darkly staining but lighten towards the medial

edges. E17 &E18

A low power overview of the GAP 43 labelled spinal cord shows that the dorsal columns have expanded further medially so that they practically merge. The white matter has established collaterals in the grey matter from the lateral column and to a greater extent the ventral column. The ventral commissure is no longer labelled. On closer examination the dorsal horn contains many fme-diameter longer collaterals that emanate from a more medial aspect of the dorsal columns. These travel in a ventrolateral arc to the dorso-lateral funiculus, which is more faintly labelled than the rest of the lateral column. There are long collaterals that emerge from the most medial aspect of the dorsal columns then grow towards the ventral horn. The dorsal horn is also characterised by many clumps of staining, these are largest medially but more discrete and numerous in the superficial and lateral aspect. There is little evidence of labelling in the ventral hom.

A day later at E l8 , the dorsal columns have enlarged and moved ventrally to form

the dorsal horns proper. The dorsal-lateral funiculus is as fully stained as the rest of the lateral column and there is increased invasion of the grey matter from the lateral columns, and is extensive from the ventral columns. In the dorsal hom, ventrally directed collaterals originating from the medial dorsal columns are not as evident. Afferents to the lateral dorsal column grow deeper than at E17 and there is increased clumps of staining in the superficial dorsal horn.

PGP 9.5 is not as revealing over these 48 hrs as the GAP 43 marker. The differences observed are primarily in the dorsal horn. The dorsal columns are not as

grey consists of a general network of fibres but clearly directed lateral or ventral collaterals are not observed. Intrusion of white matter collaterals to the grey matter only begins from the ventral column at E l8 . However one unique feature at B17 was the

presence of some fibres dorsal to the central canal which had disappeared by BIS and alsosome cellular labelling extending ventrally from the most dorsal part of the ventral horn.

Peripherin labelling also shows some unique features. As with the other markers, the dorsal columns have merged but at BIS these have not moved as far ventrally. Also, continuing the pattern set at B15 with this antibody, the dorsal horn collaterals have still not materialised in the most lateral aspect, which is inconsistent with all other labels that begin growth in a lateral to medial progression. At B17 afferents arising from the medial dorsal column grow ventrally straight to the motoneuron pool and have reached the deepest laminae. There are no laterally orientated fibres. Slightly medial to these fibres are some shorter collaterals also aimed ventrally. By BIS, these have grown deeper into the dorsal hom and appear to move laterally whilst the very long axons destined for the motor pool are no longer detectable in the deeper laminae. The disappearance of collaterals bound for the motor pool coincides with decreased cellular staining in this region at BIS. Previously at B17, there was a transient appearance of numerous very intensely labelled cell bodies with short fibres below them, directed ventrally. Their appearance was during the period when the dorsal hom axons were directed towards this target. However at BIS, these cell bodies have diminished in intensity and number and retracted ventrally, in conjunction with the presence of the fibres between the motor pool and the ventral root. Another curious feature is the lack of labelling in the white matter. The only part where labelling occurs at B17 and BIS is at the ventral root exit zone.

RT97 staining changes little between B17 and BIS but exceptions to the above descriptions exist. The pattern of labelling in the dorsal hom resembles peripherin as the lateral dorsal horn is devoid of afferents, this empty area has increased in size since first observed at B 16, however, unlike peripherin this area Was occupied by RT97 collaterals at an earlier stage of in-growth. Also at B17, similar to GAP 43, there are many clumps of

staining in the lateral superficial dorsal horn. Collaterals leaving the medial dorsal horn have a trajectory which leads them ventrally, then laterally to the lateral column. More projections cross the intermediate grey matter and are directed medially and dorsally towards the central canal. By E l8 the axons emanating from the medial dorsal hom

diverge into two separate populations. Both initially move ventrally then the first population turns laterally in the region of the dorsal-lateral funiculus. The second continues projecting ventrally towards the motor pool. This pattern is complicated considerably by the arrival of axons from the white matter and from projections crossing the intermediate grey matter from the most lateral aspect of the deep dorsal hom. These project towards the central canal and on a few occasions appear to cross-over to the contra-lateral side. In the ventral hom there is still a large population of afferents that have ramified extensively throughout this area. The most notable feature in this region is a small intensely stained group of medially positioned fibres that project ventrally and join the ventral commisure to cross ventromedially through the grey matter.

Of the small fibre markers, there is still no labelling with SP at E17 and CGRP spinal cord labelling is restricted to the motoneuron cell bodies. These cells are very dense and positioned in the dorsal region of the ventral horn and have moved ventrally at E l8 .

Also at E l8 , some fine SP fibres with a beaded appearance originate from the medial

dorsal columns, these sweep in a ventro-lateral arc to the lateral columns. The heaviest labelling which also appears to be cellular is situated near the midline in the deeper dorsal horn.

The first labelling of the superficial dorsal hom corresponding to future nociceptive fibres is observed between E17 and E l8 . At E l 7, trkA labelling of the dorsal columns has

increased since E l6 and the first in-growth of trkA afferents to the spinal grey matter

occurs. Fine diameter afferents enter from the lateral dorsal columns and reach lamina I and

n.

Within 24 hrs these axons have increased in number and extended deeper but are still confined to superficial dorsal horn, while shorter axons are observed originating from

extent of the dorsal columns, protruding ventrally into the superficial dorsal hom. These are fine but numerous, producing a network of fibres.

E19, E20 & E21

Individual paths of axons are difficult to define in the E19 GAP 43 labelled spinal cord. More noticeable is the large clumped staining in the deep medial dorsal horn. Labelled fibres are found spanning the intermediate grey matter, these cross to the contralateral side. Dense new labelling is also found in the superficial and lateral dorsal horn. There is general background staining in all of the ventral horn. At E20, labelling across the intermediate grey matter is intensified, this may be due to invasion of the white matter that crosses contralaterally. In addition, the dorsal horn is now heavily labelled by a network of fibres, this is more distinct and especially dense in the superficial dorsal horn. Only a few clumps of staining remain, these are positioned immediately lateral to the central canal.

PGP 9.5 produces overall staining of the spinal cord which makes it difficult to identify individual axons since staining is mostly cellular. At E19 however, there is clearly much heavier labelling in the superficial dorsal hom.

The changes at E19 with peripherin are restricted to the dorsal horn where three distinct groups of labelling are detected. The most striking of these are the collaterals arising from the medial dorsal columns as described at E l 7. They have not reached the ventral hom since E18 but still persist, projecting ventrally in the dorsal hom. The second group also arise from the medial dorsal columns but these project ventro-laterally, terminating at the dorso-lateral funiculus and the tangle of axons occupying the superficial dorsal horn. The final group are first detected at this age and consist of short collaterals restricted to laminae I and II and the lateral part of the dorsal hom, a location where no previous labelling with peripherin was identified. There are also some faint fibres corresponding to those of GAP 43 crossing through the intermediate grey to the contralateral side.

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Few changes are observed with RT97 except that fibres in the intermediate grey matter are less intensely stained.

CGRP labelling shows many changes at E l9. A small number of afferents enter the grey matter of the lateral dorsal horn. These project ventrally and are restricted to the lateral region. Another group of fibres arise just medial to the first group, these move further medially before turning ventrally. There are also one or two faintly stained axons that appear from the medial dorsal column and extend a short distance ventro-medially. These have a beaded appearance with axons from both sides meeting dorsal to the central canal. In the ventral horn there are a few darkly stained cells bilaterally in the ventro-medial portion.

SP cellular labelling at E19 has moved laterally from its location at E l8 , and is

now located deep in the mid-dorsal hom. TrkA labelling also develops further at this age. The superficial laminae of the dorsal horn is completely covered by a dense network of fibre and cellular labelling. There are a few lightly stained afferents leaving the medial dorsal column that move ventrally and medially extending to lamina II.

In document Análisis de los factores que influyen sobre el desempleo juvenil en el Distrito de Pichacani – Laraqueri de la Región Puno, año 2019 (página 34-39)