viernes, 27 de septiembre de 2013

In vitro regeneration of sympathetic neurons

In this article, researchers show that, although previous findings suggested otherwise, a conditionig lesion favours neuron regeneration (measured as the sprouting of new neurites) in vitro. These results were proved in mice and rats. These findings and their implications will be discussed in here.

Conditioning lesions:

A conditioning lesion consists of creating axonal damage some time before axotomy (also called "test lesion"). It has already been proven in vivo that a conditioning lesion enhances neuron regeneration after an axotomy, both in sensitive neruons and in motoneurons, being much easier to appreciate in sentitive neurons.

Methods:

Researches used the same approach to operate both mice and rats. Male adult mice (C57BL/6J) and male adult (both of them are 8 weeks old) Sprague-Dawley rats. Both models were operated (conditioning lesion) one week before the operation in which their Superior Cervical Ganglion (SCG) was sectioned and extracted. After this operation, SCGs from both mice and rats were cultured in vitro, and the results, discussed..



The present work examined the size of neurites in the explanted ganglia, contrasting results in rat and mice, and between Matrigel-cultured explants (predominantly laminin, was supposed to perform better) and collagen-cultured explants (were supposed to not enhance neurite outgrowth so much). Finally, they assessed the role of LIF (Leukemia inhibitory factor), which was supposed to be necessary for neurite outgrowth. For this part, they used LIF-null mice and LIF wt mice, finding no differences in neurite outgrowth among the two models.

One or two weeks before explantation, conditioning lesions were performed in SCG. Rats and mice were divided in two groups:
-One group with unilateral axotomy (proximal injury, transaction of ECN and ICN –external and internal carotid nerves-)
-One group with unilateral sialectomy (distal injury, salivary gland removed unilaterally)
The contralateral SCG in both of these groups, which were not modified, were taken as shams for this experiment.

Conditioning lesions were also performed, as described above, in mice homozygous for a mutation on the LIF gene, one week prior to explantation or dissociation.


SCG explantation:
One or two weeks after the conditioning lesion, animals were sacrificed by CO2 inhalation, and SCG were removed, desheathed (taken out of their sheath, of their cover), and explanted onto culture plates with either matrigel or collagen gel. Phase-contrast images of neurite outgrowth from each SCG were captured at 6, 12, 18, 24, 48 and 72 h after explantation, using an Axiovert 405 M microscope (10x magnification). After imaging at 48 or 72 h., SCG were fixed in 4% PFA (parafolmaldehyde) for 1h.

Immunohistochemistry:
After fixation, SCG were labelled with mouse anti-BIII tubulin mAB, which immunoreactivity for BIII tubulin was detected by a secondary antibody: Cy-3 conjugated donkey anti-mouse IgG. This immunohistochemistry assay was performed to verify that the processes observed in explanted SCG were neurites.


(The article goes on, but the idea was resuming one article per day, and I have already spent 2 days with this one, so I will leave it here, at least for now.)

Source: Shoemaker SE, Hyatt Sachs H, Vaccariello SA and Zigmond RE. A conditioning lesion enhances sympathetic neurite outgrowth (added by me: "in vitro"). Experimental Neurology, 2005. 194; 432-443.

miércoles, 25 de septiembre de 2013

Wallerian degeneration

This article is part of a series created to help myself learn Neurobiological concepts and develop my scientific writing. I intend to write one entry everyday, or if not, at least 3 a week. My objective is to make an interesting reading, comprehensible, but scientifically accurate (I don't want to delve so much into a "divulgating style" that it wouldn't be interesting for a biology student). I hope you like my project!

Wallerian degeneration

Wallerian degeneration is the process of axon degeneration, distal to a site of transection. It can take place in the peripheral nervous system (PNS) and in the central nervous system (CNS), but the process is different in each of these systems.

PNS: After a peripheral nerve transection, the distal axon starts to degenerate. Macrophages come into the nerve and remove myelin and axonal debris. The Schwann cells and the basement membrane around the axon remain there, but Schwann cells undergo a change in their shape, which allows them to start secreting neurotrophic factors, that favour axon growth. The basement membrane and the Schwann cells are creating "tunnels" that go directly to the muscle that has lost innervation. Axons proximal to the injury site might start sending axonal growth cones that are attracted to Schwann cells-secreted neurotrophic factors. If these axons enter one of the aforementioned "tunnels", then they will probably be capable of arriving to the target muscle, reinnervating it. Although, this reinervation is not usually done in the same way that it was before, and therefore, regeneration of peripheral nerves might entail a certain loss of function (because of a motoneuron innervating a wrong group of muscle fibers -too big or too small for it-, because of a bad growth, because of insufficient reinervation,...) or other related conditions. After this axonal growth, Schwann cells remyelinate these axons; the newly formed axons can be recognized for having a thinner myelin cover, and shorter-than-usual internodes.

CNS: Wallerian degeneration in the CNS can be seen in spinal cord trauma -I guess Wallerian degeneration is by definition possible in the brain (there are also axons in the brain), but I've got the feeling that it is just applied to the spinal cord; probably because in the brain, proximal and distal terms are not so clear; anyway, a Wallerian degeneration in the brain would probably look quite different than Wallerian degeneration after spinal cord trauma-. Spinal cord injure courses with: 1) a contusion/bruise at the site of injury, 2) a cone of hemorragic necrosis in the central section of the cord, extending several centimetres above and below the site of injury, 3) Wallerian degeration of the axons distal to the site of injury: macrophages remove debris for 2-4 weeks, and astrocytes (GFAP positive cells), proliferate largely and cover the distal axons, 4) this "astrocyte scar" is thought to be guilty of the loss of capacity of spinal cord nerves to regenerate, the GFAP scar avoids axon regeneration into the distal section.

Source: http://missinglink.ucsf.edu/lm/ids_104_cns_injury/response%20_to_injury/walleriandegeneration.htm


Three different types of lesion of the peripheral nerve:
- Neuropraxia or axonopraxia: interruption of neural conduction without proof of nerve damage. There is no Wallerian degeneration.
- Axonotmesis: only the axons are injured, and connective sheats of the nerve are preserved (endoneurium, perineurium and epineurium). Regeneration is common and functional recovery is usually good.
- Neurotmesis: endoneurial tubes loss their continuity. Repair is possible, but aberrant connectivity after the injury is common. In this case, axon-target reconnection is not specific, and thus, it is usually incorrect.

Sources: http://es.wikipedia.org/wiki/Lesi%C3%B3n_de_nervio#Axonotmesis and the article wich this entrance is based at: Allodi I, Udina E and Navarro X. Specificity of peripheral nerve regeneration: Interactions at the axon level. Prog in Neurobiol. 2012; 98: 16-37.