An atomic force microscope operating at hypergravity for in situ measurement of cellular mechano-response.

We present a novel atomic force microscope (AFM) system, operational in liquid at variable gravity, dedicated to image cell shape changes of cells in vitro under hypergravity conditions. The hypergravity AFM is realized by mounting a stand-alone AFM into a large-diameter centrifuge. The balance between mechanical forces, both intra- and extracellular, determines both cell shape and integrity. Gravity seems to be an insignificant force at the level of a single cell, in contrast to the effect of gravity on a complete (multicellular) organism, where for instance bones and muscles are highly unloaded under near weightless (microgravity) conditions. However, past space flights and ground based cell biological studies, under both hypogravity and hypergravity conditions have shown changes in cell behaviour (signal transduction), cell architecture (cytoskeleton) and proliferation. Thus the role of direct or indirect gravity effects at the level of cells has remained unclear. Here we aim to address the role of gravity on cell shape. We concentrate on the validation of the novel AFM for use under hypergravity conditions. We find indications that a single cell exposed to 2 to 3 x g reduces some 30-50% in average height, as monitored with AFM. Indeed, in situ measurements of the effects of changing gravitational load on cell shape are well feasible by means of AFM in liquid. The combination provides a promising technique to measure, online, the temporal characteristics of the cellular mechano-response during exposure to inertial forces.

Behavioural consequences of hypergravity in developing rats.

Gravity represents a stable reference for the nervous system. When the individual is increasing in size and weight, gravity may influence several aspects of the sensory and motor developments. To clarify this role, we studied age-dependent modifications of several exteroceptive and proprioceptive reflexes in five groups of rats conceived, born and reared in hypergravity (2 g). Rats were transferred to normal gravity (1 g) at P5 (post-natal day 5), P10, P15, P21, and P27. Aspects of neural development and adaptation to 1 g were assessed until P40. Hypergravity induced a delay in growth and a retardation in the development of contact-righting, air-righting, and negative geotaxis. However, we found an advance in eye opening by about 2-3 days in HG-P5 and HG-P10 rats and an increase in grip-time. No differences were found in tail and grasp reflexes. Our results show that hypergravity leads to a retarded development of motor aspects which are mainly dependent upon the vestibular system.

Effects of hypergravity on the morphological properties of the vestibular sensory epithelium. II. Life-long exposure of rats including embryogenesis.

Rats were exposed to a hypergravity (HG) level of 2.5 x g from conception until the age of 14 weeks. The vestibular epithelia of four of these animals and four control animals were immunohistochemically labeled for actin and tubulin. The apical cross-sectional area of epithelial cells of HG exposed rats appeared to be larger in all end organs. Area increase was 7.0% in the utricle (p<0.005) and 8.2% in the crista (p<<0.001). Hair cells and supporting cells appeared to be intact. The cellular arrangement and the proportion of different cell types within the epithelia was normal.

Effects of hypergravity on the morphological properties of the vestibular sensory epithelium. I. Long-term exposure of rats after full maturation of the labyrinths.

The effect of prolonged exposure to hypergravity on the morphology of vestibular epithelia of rats was investigated. At the age of 1 month, i.e., when vestibular end organs are fully maturated, three rats were transferred to a hypergravity environment of 2.5 g inside a large radius centrifuge. After 9 months, vestibular epithelia of these animals and of three control animals were immunohistochemically labeled for actin and tubulin. The apical cross-sectional area of epithelial cells of hypergravity exposed rats appeared to be smaller in all end organs. Area reduction was 1.9% in the saccule (not significant), 5.0% in the utricle (p < 0.005), and 11.6% in the crista (p<<0.001). No indications for a deterioration of vestibular functioning were observed.

Morphometric analysis of the vestibular sensory epithelia of young adult rat.

The appearance of vestibular sensory cells and their progressive development has been the subject of many ontogenetic studies. Because deteriorating hair cells are supposed to play a role in balance disorders of the elderly, the final stage of development (i.e. senescence) has been investigated as well. It is generally assumed that the number of hair cells in crista ampullaris, saccule and utricle slowly but steadily decreases with age. However, actual data covering the period between maturation and senescence are scarce. In the present study, rat vestibular epithelia were labeled for actin and tubulin. Morphology was inspected from immediately after weaning until the age of 12 months. Although, postnatal development was no part of this study some data on one day old epithelia are presented for comparison. At postnatal day 1, hair bundles are still shorter than in mature sensory organs, the width of the zonula adherens is less, and the apical cross-sectional area of the epithelial cells is smaller. After one month, maturation is complete. Total cell density is 400-500 per 0.01 mm2, both in the otolith maculae and in the cristae ampullares. During the first year after maturation, no changes in epithelial morphology were observed and cell density remains constant.