Study protocol: understanding the pathophysiologic mechanisms underlying delirium in older people undergoing hip fracture surgery.

BACKGROUND: Postoperative delirium (POD) is a common complication of older people undergoing hip fracture surgery, which negatively affects clinical- and healthcare-related outcomes. Unfortunately, POD pathophysiology is still largely unknown, despite previous studies showing that neuroinflammation, neuroendocrine dysfunction, increased reactive oxidative stress (ROS), and endothelial dysfunctions may be involved. There is also evidence that many of the pathophysiological mechanisms which are involved in delirium are involved in sarcopenia too. This article describes the protocol of a pilot study to evaluate the feasibility of a larger one that will explore the pathophysiological mechanisms correlating POD with sarcopenia. We will analyse whether various biomarkers reflecting neuroinflammation, ROS, neuroendocrine disorders, and microvasculature lesions will be simultaneously expressed in in the blood, cerebrospinal fluid (CSF), and muscles of patients developing POD. METHODS: Two centres will be involved in this study, each recruiting a convenient sample of ten older patients with hip fracture. All of them will undergo a baseline Comprehensive Geriatric Assessment, which will be used to construct a Rockwood-based Frailty Index (FI). Blood samples will be collected for each patient on the day of surgery and 1 day before. Additionally, CSF and muscle fragments will be taken and given to a biologist for subsequent analyses. The presence of POD will be assessed in each patient every morning until hospital discharge using the 4AT. Delirium subtypes and severity will be assessed using the Delirium Motor Subtype Scale-4 and the Delirium-O-Meter, respectively. We will also evaluate the patient's functional status at discharge, using the Cumulated Ambulation Score. DISCUSSION: This study will be the first to correlate biomarkers of blood, CSF, and muscle in older patients with hip fracture.

Calretinin and calbindin D-28k, but not parvalbumin protect against glutamate-induced delayed excitotoxicity in transfected N18-RE 105 neuroblastoma-retina hybrid cells.

Excitotoxic effects leading to neuronal cell degeneration are often accompanied by a prolonged increase in the intracellular level of Ca(2+) ions and L-glutamate-induced toxicity is assumed to be mediated via a Ca(2+)-dependent mechanism. Due to their buffering properties, EF-hand Ca(2+)-binding proteins (CaBPs) can affect intracellular Ca(2+) homeostasis and a neuroprotective role has been attributed to some of the family members including calretinin, calbindin D-28k and parvalbumin. We have stably transfected N18-RE 105 neuroblastoma-retina hybrid cells with the cDNAs for the three CaBPs and investigated the effect of these proteins on the L-glutamate-induced, Ca(2+)-dependent cytotoxicity. Several clones for each CaBP were selected according to immunocytochemical staining and characterization of the overexpressed proteins by Western blot analysis. In calretinin- and parvalbumin-expressing clones, expression levels were quantitatively determined by ELISA techniques. Cytotoxicity of transfected clones was quantified by measurement of the activity of lactate dehydrogenase (LDH) that was released into the medium after L-glutamate (10 mM) exposure as a result of necrotic cell death. In untransfected and parvalbumin-transfected cells, LDH released into the medium progressively increased (starting from the 20th hour) reaching maximum levels after 28-30 h of glutamate application. In contrast, LDH release in both, calretinin and calbindin D-28k-transfected clones, was not significantly different from unstimulated transfected or untransfected cells over the same period of time. The results indicate that the 'fast' Ca(2+)-buffers calretinin and calbindin D-28k, but not the 'slow' buffer parvalbumin can protect N18-RE 105 cells from this type of Ca(2+)-dependent L-glutamate-induced delayed cytotoxicity.

Calretinin and calbindin D-28k delay the onset of cell death after excitotoxic stimulation in transfected P19 cells.

In some neurological diseases, injury to neurones reflects an over-stimulation of their receptors for excitatory amino acids. This response may disturb the Ca(2+)-homeostasis and lead to a pronounced and sustained increase in the intracellular concentration of this ion. On the basis of data derived from correlative studies, calcium-binding proteins have been postulated to play a protective role in these pathologies. We tested, directly, the capacity of the three calcium-binding proteins calretinin (CR), calbindin D-28k (CB) and parvalbumin (PV) to buffer [Ca(2+)], and to protect cells against excitotoxic death. We used P19 murine embryonic carcinoma cells, which can be specifically induced (by retinoic acid) to transform into nerve-like ones. The differentiated cells express functional glutamate-receptors and are susceptible to excitotoxic shock. Undifferentiated P19-cells were stably transfected with the cDNA for CR, CB or PV, induced to differentiate, and then exposed to NMDA, a glutamate-receptor agonist. The survival rates of clones expressing CR, CB or PV were compared with those of untransfected P19-cells using the lactate-dehydrogenase assay. CR- and CB-expressing cells were protected from death during the first 2 h of exposure to NMDA. This protection was, however, transient, and did not suffice to rescue P19-cells after prolonged stimulation. Two of the three PV-transfected clones raised were vulnerable to NMDA-induced excitotoxicity; the third, which expressed the lowest level of PV, was protected to a similar degree as that found for the CR- and CB-transfected clones. Our results indicate that in the P19-cell model, CR and CB can help to delay the onset of cell death after excitotoxic stimulation.

Calretinin-immunoreactivity during postnatal development of the rat isocortex: a qualitative and quantitative study.

Postnatal development of the rat cortex is characterized by the gradual development of many calcium-dependent processes which demand a precise control of the intracellular levels of this cation; when the balance is disturbed, neuronal death ultimately ensues. Calretinin (CR), a calcium-binding protein, has been postulated to have neuroprotective capacity by buffering intracellular calcium. This putative relationship between CR and neuroprotection is still, however, a controversial issue. With a view to shedding further light on this subject, we studied the temporal and spatial distribution of CR in five different regions (the frontal- sensorimotor-, parietal-, temporal- and occipital region) of the rat cortex during postnatal development. Qualitative and quantitative immunocytochemistry of newborn, 5-, 10-, 15-, 20- and 30-day-old and adult rats revealed a profound increase in the density of the CR-positive neurons during the first two postnatal weeks in all regions examined. At the end of this period, CR-immunoreactive cells decreased sharply to adult levels. Cell classes exhibiting transient CR-immunoreactivity during the first two postnatal weeks included cells in layer I (amongst which were Cajal-Retzius cells), the subplate and pyramidal-like cells in the upper portion of layer V, most of them in the motor cortices. The above-described dynamics of CR expression were reflected also in the biochemical analysis performed (immunoblotting, ELISA). The temporal and spatial correlation with calcium-dependent events such as synaptogenesis, neurite elongation and remodelling in further support the contention that CR may play a neuroprotective role during postnatal development of the rat cortex.