Study Brain 59: Optic tract healing

Findings

This rat, 571g, was perfused 18 days after a 1mm U device, held parallel to the midline and 3.5mm lateral to it, 2.5mm posterior to Bregma and aimed at the optic tract, was lowered to the floor of the brain. Since the optic tract had dramatically remodeled around the cut, coronal sections were taken of the anterior brain, including the nerves, as well as a complete horizontal series from cortex to ventral.

One optic nerve had a grossly new pattern. Instead of a parallel arrangement of thick axons, it was bordered by this (except most medially where it was absent) but in its central portion was a vertically oriented population, with new-grown features, focused on the most ventral nerve. Surprisingly, this population was more immature nearer to the eye.

Remodeling of optic tract as neurons grow around the cut

The horizontal limb of the optic tract appears to have remodeled ventrally, around lower part of the device’s arms. This exposes the hypothalamus in the plane of the section with the robust intact tract. Pictures of its patterns are in (241&5), (321&4, 332&2, 333, 341&4), (422&3, 433), (524, 535, 545) and (621&2). The unlikely alternative, that the intact tract had remodeled dorsally, is being examined by characterizing the interanimal variation of axon patterns near the optic chiasma. The most dorsal part of the AP optic tract has a new pattern at its entry into the hypothalamus divided into two populations, medial and lateral The lateral projects into the hypothalamus, The medial, denser and dark-axoned doesn’t seem to make it.

The medial forebrain (MFB) and projection to the ventromedial hypothalamus, exposed by this migration, have a dense medial detour of AP fasciculi at the cut, which is crossed by ML optic tract axons en route to/from its lateral remodeling (535). The optic tract distal to the detour has abundant optic and SOD axons, although perhaps fewer than normal, and the thin (75 um) blue portion between these has many axons also. Score 3+ The more dorsal MFB has subtle detours and rather normal populations on each side. IN (82) it seems hyper-healed, with a few thin axons crossing as well. Score 5.

The MFB, 600 um caudal to the end of the detoured cut, seems to have a normal axon population (624). Also, it passes by both sides of the cut and has fine signs of neoformation (625). Rostrally, it seems rather normal (641).

On right, massive detour of the fimbria. Left side shows intact brain.

The fornix distal to the cut that was right in its way (731, which also shows the ascending optic tract) seems normal. (825) has a view of the normal at that AP coordinate. Rostral to the device, the fornix stops its ascent and reorients dorsally, with isolated bundles and a new spiral pattern of its body-bundles. It seems to have as many axons as the intact one. In (8?023) its rostral tip bends slightly medially and it is organized more as a bundle than as a long horizontal path. However, the intact fornix is like this is (914). Comparing the two ((925) the one on the cut side seems normally organized, with darker-staining axons and blue myelin, but no indications of new growth.

(925) the cut is now through the bed n stria terminalis with a sparse caudal detour. Score 1. inferior thalamic radiation, also cut in (933), detours towards the medial ventral thalamus (933) and ventromedial thalamus (1013 P) where it also makes an anterior detour into the anterior thalamus. Take pictures here to compare the anterior thalamus on both sides, also tvm. To the anterior thalamus has a score of 4, to the TVM of 5. This will be an excellent comparison with the three month results!

stria terminalis, which was infringed, is dark-stained and has a massive detour, score 4 (1033)

fimbria, nicked most ventrally, also detours massively (1035+); it surrounds both side of the cut ( P 1123) and only has a few, lateral, axons perpendicular to it. It begins as an anterior detour of nearly all the axon population of the lateral side (the middle population, in the blue-free zone, also detours) the anterior ~1/3 of the medial cut fimbria while the more caudal cut fimbria makes a caudal detour. In (1121) the medial caudal, and rostral anterior, detours are most massive.. They are ~equally massive in (1123). ten the caudal begins to become most massive. The result is a massive filling of the fimbria (1131). score 3+ with a few bright blue areas. Score 3++. The alveus detours (1211) score 4

corpus callosum detours massively, score 4 or 4+ ( P 1222-4). The wire separation is only 550 um now, so there was unmarked cut here.

cortex, anterior and posterior to the cut, when compared with the corresponding intact side may seem uncut (1613). It is ambiguous because the actual cut was longer than the hole separation.

Method

This male Long Evans rat, probably a retired breeder donated by Curt Richter’s lab and transported from the Med School by me in a box, weighed 570g. A U-shaped device, ~1mm wide and positioned parallel to the midline, was held in curved forceps (probably capsule forceps which hold well and release well). The anterior end was positioned 3.5mm lateral to the midline as defined by Bregma, the intersection of the anterior suture lines, and 2.5mm posterior to Bregma. These planes were defined by the orthogonal scalpel-cuts I had made on the skull, under the dissecting microscope, and filled with soft pencil lead. I oriented the device to be perpendicular by referring to an insect pin nearby, held in movable jaws, which was made vertical by sighting along two plumblines made of fine suture weighted by a little triangular plastic container made by cutting off the corner of a Baggie and filling it with copper shot.

I made an antero-posterior burrhole with a fine dental drill, beginning at the planned entry of the device, and lowered the hand-held device under microscope control until either one or both of the arms showed an arrest (indicating the arrival of the crosswire at the skull) by curving slightly outward or inward. Then I released the forceps and cut off the parts of the wires protruding from the skull. This was a critical part of the procedure, because any extra movement of the device would complicate the interpretation of the results. I used clippers, scissors, etc, during the time of using this non-cemented approach, and finally settled on one pair of stitch scissors, made by Miltex, which were pretty reliable. No other tested stitch scissors, even by Miltex, worked as well.

It turned out that the post-lesion slips, etc, produced secondary cuts which, when examined immediately post-lesion, were straight and interrupted the tissue without deforming it, leaving jagged edges. Later, there was dramatic reorientation of patterns following these edges. Now, for this step, I would recommend ligature scissors made of tungsten carbide stainless steel. After cutting I placed a piece of Micropore tape over the burrhole and sutured the skin.

I’m not sure when I began routinely using tetracycline powder on the hole before putting on the Micropore tape. When there were mastoid infections, seen when I removed the ventral skull, I made a note of this. Windle’s Piromen, made from pyrogenic bacteria and producing experimental results indicating spinal cord regeneration, was on my mind here.

In the early experiments the rats were housed individually in a rack of metal cages. Later I began keeping them in the lab, in large cages with company, and giving then toys e.g. PVC pipe and wooden alphabet blocks, to provide a better healing environment (I hoped).

After 18 days, weighing 529g, he was perfused with saline followed by AATCA (1% ammonia in 95% alcohol containing 5% trichloroacetic acid). I developed this fixative, instead of the ammoniacal alcohol Ranson used in his improvement of Cajal’s block silver stain, by adding a pyridine extraction before impregnation, to make it able to show the fine c-fibers ascending the cat spinal cord, because just ammoniacal alcohol produced great shrinkage which resulted in trouble with the paraffin getting into the brain. Also, I thought that TCA might improve the counterstaining by precipitating the proteins better, since in Jim Watson’s lab at Harvard, isolating ribosomes, I used it thus. When tested by immersing pieces of fresh cerebellum if it, in a 10ml graduate to find their volume, it proved to produce no shrinkage.

Detail, remodelling of optic tract

Careful removal of the ventral skull, with the device in place, revealed a dramatic reorganization of the optic tract around the cut. After the pyridine extraction and washing steps, I withdrew the device. The anterior brain was sectioned coronally, to see the optic nerves. The remainder was sectioned horizontally, and a complete series of sections containing the optic tract was counterstained. The rest of the brain was mounted every tenth section.

Optic Nerves

Rather than the usual alignment of optic axons parallel to the nerve, that on the cut side had a new shape (more oval) and fiber pattern (a large immature-looking parallel-fibered component oriented vertically, towards a point on the central ventral nerve, and a smaller most medial one, parallel the medial surface and oriented to a ventromedial location on the nerve). These axon populations were had more immature features near the eye than ~160um caudal to this; the nerves were also smaller (Slide1).

Summary of brain 59