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.