Investigation of microbial growth in vivo: evaluation of a novel in vivo chamber implant system.

An intraperitoneal chamber implant system has been used to investigate the phenotype of Staphylococcus aureus growing in the rat and the effect of the antibiotic flucloxacillin on bacterial growth in vivo. Titanium chambers were implanted in the peritoneum: a period of 3-4 days equilibration allowed diffusion of host proteins into the chamber fluid prior to inoculation with bacteria. S. aureus inoculated into the chamber fluid, grew rapidly over a 72 h period, reaching counts of > 10(9) per ml. Organisms harvested from chambers were analysed by SDS-PAGE and showed significant differences in polypeptide profiles from the same strain grown in nutrient broth in vitro. Analysis of whole cell extracts by Western-blotting revealed that protein A expression was repressed in S. aureus grown in vivo. Following subcutaneous administration, flucloxacillin levels in serum peaked earlier and were higher than those detected in chamber fluid. The inhibitory effect of the antibiotic on the growth of S. aureus in chambers in treated animals could be monitored easily by sequential sampling of the chamber fluid. These results indicate the potential of the chamber implant model for investigation of microbial phenotype in vivo and development of alternative methods for assessment of antimicrobial efficacy in vivo.

Diversity and homogeneity within endplates associated with physiologically identified static gamma-axons in cat tenuissimus muscle.

The terminations of static gamma-axons on chain fibres may be associated with a muscle surface contour thrown into complex folds at one extreme, ranging through lesser degrees of folding to being apparently uninfluenced by the presence of the terminals. The folding is not necessarily confined to the postsynaptic membrane. As a quantitative indicator of the degree of folding seen in cross-sections, the perimeters of the two halves of the intrafusal fibre with, and without the ending were compared. Both complexity of endplate structure and an undifferentiated appearance could co-exist within individual endings (of six static gamma-axons on thirteen chain fibres), associated with axons that supplied only chain fibres in the spindle isolated. (It should be noted, however, that the type of intrafusal fibres innervated by a particular axon was definitively identified only for the spindle isolated.) Endings of three other static gamma-axons on six chain fibres had a more homogeneous and less complex endplate structure; these supplied bag2 fibres in addition to the chain fibres and their endplates on the bag2 fibres were less folded than those of three axons which supplied bag2 fibres only. A sensory inhibitory influence on folding was not apparent because complexly folded endplates on chain fibres lay close (less than 500 microns) to sensory endings. All the endings studied were functional. In our experimental conditions all nine static gamma-axons which innervated chain fibres, alone, or together with a bag2 fibre, drove the Ia afferent at some stimulus frequencies; none of the three static gamma-axons innervating bag2 fibres alone caused driving at any frequency. These findings are discussed in relation to the concept of a dynamic remodelling of ending structure during life, to the relationship between the motor axon and the intrafusal fibre it innervates, and to the possibility that subgroups of static gamma-motoneurones might exist which could release different amounts of the trophic substances responsible for moulding the endplate structure.

The selectivity of fusimotor innervation in muscle spindles of the rat studied by light microscopy.

Six muscle spindles and three muscle spindle poles from four rat soleus muscles have been sectioned serially at 1 micron intervals to trace the motor innervation by light microscopy. Forty myelinated axons had 92 endings on the intrafusal muscle fibres. 67.5% of these axons supplied a single type of muscle fibre only, 22.5% to dynamic bag1 (Db1) fibres, 15% to static bag2 (Sb2) fibres and 30% to chain fibres. The rest supplied more than one fibre type, 5% supplying the Db1 and one chain fibre, 20% supplying the Sb2 and chain fibres, but 7.5% (three axons) supplied all the fibre types together. Apart from these three axons all the fusimotor axons would be expected to have a clear dynamic or static action on the Group Ia discharge. Whilst for the cat entirely non-specific distribution does not exist, or at least is very rare, since only a small proportion of the rat fusimotor axons were in this category we conclude that fusimotor distribution in rats and cats is essentially similar.

The ultrastructure of cat fusimotor endings and their relationship to foci of sarcomere convergence in intrafusal fibres.

1. Six muscle spindle poles, five from experiments in which foci of sarcomere convergence had been observed during stimulation of fusimotor axons, were serially sectioned for light and electron microscopy. Every somatic motor terminal was studied in ultrathin sections at several levels.2. In all six poles static gamma axons, or presumed static gamma axons, supplying the static bag(2) fibre and/or chain fibres had no terminations on the dynamic bag(1) fibre. In five poles, the dynamic bag(1) fibre was selectively innervated by dynamic gamma or beta axons save in one case where a dynamic gamma axon also innervated one chain fibre.3. Seventy-seven motor endings were of four distinct ultrastructural types: ;m(a) plates' lay superficially on the surface of static bag(2) or chain fibres; ;m(b) plates' were deeply indented into dynamic bag(1) fibres; in ;m(c) plates', found on chain fibres only, the muscle surface was thrown into projecting fingers between which the axon terminals were embedded; one type ;m(d) plate' was found, fully indented into a long chain fibre. A few plates of intermediate form (m(ab)) were variants of m(a) and m(b) plates.4. The muscle membrane beneath both m(a) and m(b) plates was smooth, or had a few wide, shallow folds; m(c) plates usually had wide, shallow subjunctional folds; numerous deep, narrow folds were characteristic of the m(d) plate. The length of unmyelinated pre-terminal axon or the number of sole plate nuclei were not useful diagnostic features.5. Obvious foci of sarcomere convergence in the capsular sleeve region of dynamic bag(1) and static bag(2) fibres coincided with the location of motor plates. Additional contraction foci were observed in the extracapsular region of dynamic bag(1) fibres where there was no motor innervation; contraction occurs principally in the outer half of these fibres. No foci of contraction or motor plates were observed in the extracapsular region of static bag(2) fibres; contraction in these fibres is typically mid-polar.6. In some poles local contraction of chain fibres centred on the location of m(c) plates. In others, very localized contraction occurred distal to the sites of m(a) plates. Both m(a) and m(c) plates were never found on the same pole of a chain fibre.7. Dynamic gamma or beta axons end in m(b) plates, probably equivalent to p(2) plates. The concept of distinctly different p(1) and p(2) plates on dynamic bag(1) fibres, supplied by dynamic beta and gamma axons, respectively, is not supported by ultrastructural evidence.8. Some static gamma axons end in multiple m(a) plates which correspond with ;trail endings', or in single large m(a) plates, on static bag(2) or chain fibres. The m(c) plates are the terminations of other static gamma, or occasionally dynamic gamma, axons on chain fibres. Static beta axons probably end in m(d) plates on long chain fibres which may correspond with p(1) plates.9. It is proposed that there are two types of static gamma motoneurone, one terminating in m(a) plates and the other in m(c) plates, possibly directed preferentially towards static bag(2) fibres and chain fibres, respectively.

Ultrastructural dimensions of myelinated peripheral nerve fibres in the cat and their relation to conduction velocity.

1. The ultrastructure of all the afferent fibres, or all the efferent fibres, was studied in selected nerves from chronically de-afferentated or de-efferentated cat hind limbs perfusion-fixed with glutaraldehyde.2. The following parameters were measured: number of lamellae in the myelin sheath (n), axon perimeter (s), external fibre perimeter (S), axon cross-sectional area (A). Fibres were allocated to afferent groups I, II, III or efferent groups alpha and gamma according to the number of lamellae in the myelin sheath.3. The thickness of the myelin sheath (m) was linearly related to axon perimeter within the range s = 4 mum to s = 20 mum (groups II, III and gamma). The relation m = 0.103 s - 0.26 provided a good fit for all afferent and efferent axons in this range in several different anatomical muscle nerves in three cats. The myelin sheaths were thinner in a fourth, presumably younger, cat.4. The myelin sheaths were relatively thinner for large fibres in groups I and alpha (s = 20-50 mum). The results are interpreted in one of three ways. Either m tends to a limit of about 2.2 mum, or m is linearly related to s such that for large fibres m = 0.032 s + 1.11.5. Alternatively, m may be considered to be proportional to log(10)s for all sizes of axon so that m = 2.58 log(10) S - 1.73. The interpretation that there are two separate linear relations for large and small fibres is favoured.6. The ratio of axon to external fibre perimeter (g) falls from about 0.70 for group III and small gamma fibres in the cat to about 0.62 for group II and large gamma fibres and then rises again to 0.70, or even 0.75 for group I and alpha axons.7. The above relations between m and s are combined with the observations of Boyd & Kalu (1979) that Theta = 5.7 D for groups I and alpha and Theta = 4.6 D for groups II, III and gamma. It is shown that Theta = 2.5 s approximately for all sizes of axon (s from material fixed for electron microscopy) in rat, cat and man. The accuracy of this equation may be improved by deducting 3 m/sec in the case of small fibres. This conclusion is compatible with experimental observations of the relation between l and D (Hursh, 1939; Lubinska, 1960; Coppin, 1973) and between l and Theta (Coppin & Jack, 1972).8. From the theoretical analyses of Rushton (1951) and others Theta should be proportional to the external dimensions of the fibre rather than to axon size. It is shown that the thinning of the myelin sheath ought to affect Theta substantially. Thus some other factors must compensate for the thinning of the sheath.9. Small fibres are significantly more non-circular than large fibres. From the quantitative data of Arbuthnott et al. (1980) it is concluded that non-circularity may contribute to the fact that Theta proportional, variant s rather than Theta proportional, variant S, but cannot wholly account for it. Other possibilities considered are that axoplasmic resistivity or specific nodal conductance may differ for large and small fibres.10. It is suggested that myelinated peripheral nerve fibres may fall into two distinct classes with different properties, one comprising groups I and alpha and the other groups II, III and gamma. The conclusion predicted from theory may apply to each of these classes separately so that Theta = 2.0 S for the large-fibre class and Theta = 1.6 S for the small-fibre class.

Quantitative study of the non-circularity of myelinated peripheral nerve fibres in the cat.

1. One hind limb of each of four cats was either chronically de-efferentated, or chronically de-afferentated, and perfused with buffered glutaraldehyde fixative. Up to three different muscle nerves were dissected from each limb, post-fixed in osmium tetroxide and embedded in Epon. Ultrathin transverse sections were mounted on Formvar-coated single-hole specimen grids so that all the fibres in each nerve could be examined individually by electron microscopy.2. Non-circularity was expressed as the ratio (ø): [Formula: see text] The degree of non-circularity of all the afferent axons, or all the efferent axons, in each muscle nerve was determined. The proportion of fibres cut through the paranodal region, or through the Schwann cell nucleus, was as expected for group I afferent and for alpha and gamma efferent fibres, but hardly any typical paranodal sections of group II or III afferent fibres were encountered which suggests that their paranodal arrangement differs from that of other groups. In a quantitative comparison of noncircularity in different functional groups, fibres cut through paranodes, Schwann cell nuclei or Schmidt-Lanterman clefts were rejected.3. All the gamma efferent fibres in one nerve were studied in a series of sections cut at 25 mum intervals. The degree of non-circularity was found to be relatively constant along the internode of most fibres when the values at paranodes, Schwann cell nuclei or Schmidt-Lanterman clefts were ignored.4. The value of ø varied widely from 1.0 (circular) to 0.5 or less from fibre to fibre within every functional group. However, the mean value of ø was less for gamma axons (0.68) than for alpha axons (0.78), and less for group III axons (0.79) than for axons in groups I and II (both 0.84). When the results for all the nerves were aggregated, these differences were statistically very highly significant, as was the difference in ø between group I and alpha fibres. If values of ø < 0.5 were rejected, the difference between the mean ø for group III and group II was then of doubtful significance whereas that between alpha and gamma fibres was still very highly significant.5. The external perimeter (S) of a non-circular fibre differs from pi times the diameter of a circle just enclosing the fibre (D). It is shown that S = 0.95 pi D for group I and II fibres, S = 0.90 piD for alpha and group III fibres, and S = 0.85 piD for gamma fibres.6. The myelin period, or interperiod repeat distance, varied from 14.1 to 15.6 nm in different cats, implying radial shrinkage of the myelin sheath from 15 to 23%. The myelin period in a particular cat was the same for several nerves, and the same for fibres in different functional groups.7. The possibility that repetitive firing of axons during fixation contributed to the varying degree of non-circularity is considered but rejected as unlikely.8. It is deduced that about 10% radial shrinkage of the myelin sheath, but little or no osmotic shrinkage of the axon, occurred during fixation and rinsing. Further radial shrinkage of about 8% in all components of the fibre probably occurred as a result of subsequent histological processing. It is concluded that the non-circularity of all axons, and the greater non-circularity of small axons, is unlikely to have been due to histological processing.9. It is concluded that axons are non-circular in vivo. The hypothesis that non-circularity allows axons to accommodate swelling during repetitive activity is discussed. Suggestions are made as to why gamma axons may be more non-circular than alpha or group III axons in an anaesthetized cat immediately prior to fixation, and why alpha axons may be more non-circular than axons in groups I and II.