Practical methods for preparing demyelinated human axons

4.1 Method used fo r the first recordings

The preparations for the first successful patch clamp recordings were made as follows:

Fascicles were removed from the epi- and perineurium (section 3.2, page 46), split longitudinally to ensure complete removal of the perineurium (section 3.4.1), and tied at each end with sewing thread. The threads were pinned to the base of the incubation chamber described in section 3.3, and after adding the appropriate enzyme solution, the incubation chamber was placed in the shaking waterbath mentioned in section 3.3.

They were treated with the following enzyme mixtures:

Stage 1

Collagenase, Sigma type V (480 U/mg), 2 mg/ml (960 units/ml) Protease, Sigma type X (43 U/mg), 0.03 mg/ml ( 1.29 units/ml) Ringer with 0.1 mmol/1 [Ca^^ ]

Temperature 37°C Agitation 150/min Time 2 hours

Stage 2

Protease, Sigma type XXIV (14 U/mg), 0.5 mg/ml (7 units/ml) Trypsin, Sigma type I (10000 U/mg), 1 mg/ml (10000 units/ml) Ringer with no added Ca^"^

Temperature 37°C Agitation 150/min Time 30 minutes

Fluid flow from a large Pasteur pipette was used to spread the fascicle apart, then, after cutting it into sections approximately 5 mm long, single nerve fibres were separated from the fascicle by gentle circular agitation. These fibres, suspended in Ringer solution, were transferred to 35 mm plastic Petri dishes; normally about six dishes were obtained from each preparation. The dishes were at first coated with Glisseal grease (high vacuum quality. Borer Chemie AG, Zuchwil, Switzerland; Jonas et a l 1989), or polylysine (Sigma P-7890, 1 mg/ml). Because of the poor optical qualities of the Glisseal coating, especially with phase-contrast optics, and the unpredictable results obtained with polylysine, later experiments used Petri dishes

coated with a silico n e fluid o f high v isco sity (S ilic o n e fluid D C 2 0 0 /1 0 0 ,0 0 0 c s, D o w C orning), applied on top o f a very thin layer o f Sylgard. T his coatin g w as flat, and in visib le in phase-contrast op tics, and axon s stuck to it w ell. T he Petri d ish es were kept in the refrigerator for at least 1 hour after adding the axon su sp en sion , to allow the fibres to adhere to the silic o n e coating.

Figure 1

An axon prepared with the method described in section 4.1. Myelin retraction has occurred to an extent o f some 60 pm on one side of the node o f Ranvier, leaving an

apparently clean axonal

membrane (dark green) between the two retracted Schwann cells (lighter green). The node can be seen as a dark bulge in the constricted section o f the axon.

Phase contrast optics,

magnification 360x.

A number o f axons prepared with this m ethod had clean d em yelin ation , suitable for patch clam p in g (Figure I).

4.2 An improved enzyme mixture

Development of the preparation continued in parallel with the patch-clamp experiments. Most of the recordings described in later chapters of this thesis were made in preparations which had been dissociated with the following enzyme mixtures:

Stage 1

Collagenase, Sigma type XI, 2 mg/ml (about 3500 units/ml) Protease, Sigma type X, 0.1 mg/ml (about 4.5 units/ml) Ringer solution with 2.2 mmol/1 Ca^"*"

Temperature 37°C Agitation 50/minute

Time 1 - 2 hours (the time could be reduced in some cases)

Stage 2

Protease, Sigma type XXIV, 1 mg/ml (about 14 units/ml) Trypsin, Sigma type HI, 1 mg/ml (about 10000 units/ml) Ringer solution with 2.2 mmol/1 Ca^^

Temperature 37°C Agitation 50/minute Time 30 minutes

These enzyme mixtures are relatively simple, and similar in principle to those used by Jonas et a l (1989) and Safronov et a l (1993). The only important difference was that the enzyme activities needed were much higher than those necessary for Xenopus

or rat axons.

In the first stage, collagenase activity was about six times greater than is necessary to prepare Xenopus and rat axons. The added non-specific protease type X presumably has the same role as the rather high, but variable, non-specific protease impurities in the Worthington type CLS II collagenase used in Xenopus and rat; it probably breaks down the large fragments of collagen resulting from collagenase treatment into smaller pieces, and may attack the protein backbone of proteoglycan molecules. In the second stage, twice as much non-specific protease was used as is needed in rat axons, along with trypsin; both probably cause demyelination, and complete the digestion of fragments of collagen.

A good preparation generally contained about 10-30 nodes, paranodes or intemodes which could be patch clamped, a remarkably low yield considering that each fascicle (about 2 cm long) probably contained several hundred nodes before

d issociation . N odal and paranodal structure was often preserved extrem ely w ell (Figure 2, top), and enzym e-treated a xon s also had go od structural appearance when v iew ed w ith electron m icroscopy (Figure 3, next page). S o m e axons sh ow ed ex ten siv e d em yelin ation as a result o f the normal preparation procedure (Figure 2, bottom); on e such axon could even be pulled out from the m yelin sheath u sing a patch pipette, ex p o sin g m ost o f the length o f one internode. Seal form ation in these long bare internodes w as difficult, but it w as so m etim es p o ssib le to m ake patches more than 100 pm from the node.

k

Figure 2

(above) A demyelinated human axon prepared as described in section 4.2.

The axonal membrane at the node and in the paranode can be seen in some detail, and its morphology is very well preserved. Phase contrast optics, magnification 360x.

(below) A demyelinated human axon prepared in the same way. The myelin has retracted about 150 pm into the intemode, and a patch pipette is approaching a site about 55 pm from the node. Phase contrast optics, magnification 360x.

Figure 3

Electron micrograph of a cross- section through the intemode of an axon prepared in the same way. The cytoskeleton and mitochondria are well preserved, and only slight distortion o f the myelin is visible (although some sections had extensive myelin

damage). The endoneurial

connective tissue and basal lamina are completely absent. Magnification 8200x.

4.3 A note on the variability o f hum an nerves

N o o b v io u s age-related d ifferen ces in ease o f d issociation w ere seen am ong nerves from the 6 6 patients featured in this study. M ost o f the patients w ere less than 50 years old. O ne sp ecim en o f nerve w as from a 4 -m o n th -o ld baby; after en zym e treatment, dem yelin ation w as very clean , but separation o f sin g le fibres w as not easier than in nerves from adults.

C onsiderable variation w as seen in con n ectiv e tissu e density b etw een nerves from different sites in the sam e individual. C on n ective tissu e seem ed very dense in the brachial p lexu s, w h ile fa sc ic le s from ulnar or sural nerves from the sam e patient were m uch m ore easily d isso cia ted . There w as also som e variation in ea se o f d issociation b etw een adjacent fa sc ic le s from the sam e part o f the sam e nerve, w hich w as not o b v io u sly related to fa sc ic le thickness.

CHAPTER 3

PATCH-CLAMP RECORDINGS