Lars Sundstrom

Kainic acid increases the proliferation of granule cell progenitors in the dentate gyrus of the adult rat

Granule cell progenitors in the dentate gyrus of the hippocampal formation have the unusual capacity to be able to divide in the brains of adult rats and primates. The basal proliferation rate of granule cell progenitors in the adult rat is low compared with development, however, it is possible that this rate may become significantly altered under pathological conditions such as epilepsy. We have investigated whether the proliferation of granule cell progenitors is increased in adult rats in a model of temporal lobe epilepsy, by using systemic bromodeoxyuridine injections to label dividing cells. We report here for the first time that granule cell neurogenesis is increased bilaterally 1 week after a single unilateral intracerebroventricular injection of kainic acid. Bromodeoxyuridine labeled neurons increased at least 6-fold on the side ipsilateral to the kainic acid injection compared to controls, but significantly, were also increased, by at least 3-fold on the side contralateral to the injection. The dividing cells in the subgranular zone were identified as neurons since they expressed Class III beta tubulin but not glial fibrillary acidic protein.

Neuropeptide Y is neuroproliferative for post-natal hippocampal precursor cells

New neurones are produced in the adult hippocampus throughout life and are necessary for certain types of hippocampal learning. Little, however, is known about the control of hippocampal neurogenesis. We used primary hippocampal cultures from early post‐natal rats and neuropeptide Y Y1 receptor knockout mice as well as selective neuropeptide Y receptor antagonists and agonists to demonstrate that neuropeptide Y is proliferative for nestin‐positive, sphere‐forming hippocampal precursor cells and β‐tubulin‐positive neuroblasts and that the neuroproliferative effect of neuropeptide Y is mediated via its Y1 receptor. Immunohistochemistry confirmed Y1 receptor staining on both nestin‐positive cells and β‐tubulin‐positive cells in culture and short pulse 5‐bromo‐2‐deoxyuridine studies demonstrated that neuropeptide Y has a proliferative effect on both cell types. These studies suggest that the proliferation of hippocampal neuroblasts and precursor cells is increased by neuropeptide Y and, therefore, that hippocampal learning and memory may be modulated by neuropeptide Y‐releasing interneurones.

An in vitro model of traumatic brain injury utilising two-dimensional stretch of organotypic hippocampal slice cultures

Traumatic brain injury (TBI) is caused by rapid deformation of the brain, resulting in a cascade of pathological events and ultimately neurodegeneration. Understanding how the biomechanics of brain deformation leads to tissue damage remains a considerable challenge. We have developed an in vitro model of TBI utilising organotypic hippocampal slice cultures on deformable silicone membranes, and an injury device, which generates tissue deformation through stretching the silicone substrate. Our injury device controls the biomechanical parameters of the stretch via feedback control, resulting in a reproducible and equi-biaxial deformation stimulus. Organotypic cultures remain well adhered to the membrane during deformation, so that tissue strain is 93 and 86% of the membrane strain in the x- and y-axis, respectively. Cell damage following injury is positively correlated with strain. In conclusion, we have developed a unique in vitro model to study the effects of mechanical stimuli within a complex cellular environment that mimics the in vivo environment. We believe this model could be a powerful tool to study the acute phases of TBI and the induced cell degeneration could provide a good platform for the development of potential therapeutic approaches and may be a useful in vitro alternative to animal models of TBI.

Organotypic cultures as tools for functional screening in the CNS

A major challenge for the pharmaceutical industry is the development of relevant model systems in which knowledge gained from high-throughput, genomic and proteomic approaches can be integrated to study function. Animal models are still the main choice for such studies but over the past few years powerful new in vitro systems have begun to emerge as useful tools to study function. Organotypic cultures made from slices of explanted tissue represent a complex multi-cellular in vitro environment with the potential to assess biological function and are uniquely placed to act as an important link between high-throughput approaches and animal models.

7‐Hydroxylated epiandrosterone (7‐OH‐EPIA) reduces ischaemia‐induced neuronal damage both in vivo and in vitro

Recent evidence suggests that steroids such as oestradiol reduce ischaemia‐induced neurodegeneration in both in vitro and in vivo models. A cytochrome P450 enzyme termed cyp7b that 7‐hydroxylates many steroids is expressed at high levels in brain, although the role of 7‐hydroxylated steroids is unknown. We have tested the hypothesis that the steroid‐mediated neuroprotection is dependent on the formation of 7‐hydroxy metabolites. Organotypic hippocampal slice cultures were prepared from Wistar rat pups and maintained in vitro for 14 days. Cultures were then exposed to 3 h hypoxia and neuronal damage assessed 24 h later using propidium iodide fluorescence as a marker of cell damage. Neurodegeneration occurred primarily in the CA1 pyramidal cell layer. The steroids oestradiol, dehydroepiandrosterone and epiandrosterone (EPIA) were devoid of neuroprotective efficacy when present at 100 nm pre‐, during and post‐hypoxia. The 7‐hydroxy metabolites of EPIA, 7α‐OH‐EPIA and 7β‐OH‐EPIA significantly reduced neurotoxicity at 100 nm and 10 nm. 7β‐OH‐EPIA was also neuroprotective in two in vivo rat models of cerebral ischaemia: 0.1 mg/kg 7β‐OH‐EPIA significantly reduced hippocampal cell loss in a model of global forebrain ischaemia, whereas 0.03 mg/kg was neuroprotective in a model of focal ischaemia even when administration was delayed until 6 h after the onset of ischaemia. Taken together, these data demonstrate that 7‐hydroxylation of steroids confers neuroprotective efficacy, and that 7β‐OH‐epiandrosterone represents a novel class of neuroprotective compounds with potential for use in acute neurodegenerative diseases.

Granule cell dispersion is correlated with early epileptic events in human temporal lobe epilepsy

Granule cell dispersion (GCD) into the dentate gyrus (DG) molecular layer was observed in hippocampal specimens in 10 out of 22 cases of human non-lesional temporal lobe epilepsy (TLE) and was associated with hippocampal sclerosis (HS). The presence of GCD was significantly linked to events of epileptic nature arising during the first 4 years of life but not with the durations of epilepsy, nor the number of seizures. Dispersion could be induced by seizure-linked structural plasticity occurring during a specific early permissive period.

Lactate and glucose as energy substrates during, and after, oxygen deprivation in rat hippocampal acute and cultured slices

The effects of raised brain lactate levels on neuronal survival following hypoxia or ischemia is still a source of controversy among basic and clinical scientists. We have sought to address this controversy by studying the effects of glucose and lactate on neuronal survival in acute and cultured hippocampal slices. Following a 1‐h hypoxic episode, neuronal survival in cultured hippocampal slices was significantly higher if glucose was present in the medium compared with lactate. However, when the energy substrate during the hypoxic period was glucose and then switched to lactate during the normoxic recovery period, the level of cell damage in the CA1 region of organotypic cultures was significantly improved from 64.3 ± 2.1 to 74.6 ± 2.1% compared with cultures receiving glucose during and after hypoxia. Extracellular field potentials recorded from the CA1 region of acute slices were abolished during oxygen deprivation for 20 min, but recovered almost fully to baseline levels with either glucose (82.6 ± 10.0%) or lactate present in the reperfusion medium (108.1 ± 8.3%). These results suggest that lactate alone cannot support neuronal survival during oxygen deprivation, but a combination of glucose followed by lactate provides for better neuroprotection than either substrate alone.

Bilateral reorganisation of mossy fibres in the rat hippocampus after a unilateral intracerebroventricular kainic acid injection

One month after a unilateral intracerebroventricular injection of the neurotoxin kainic acid (KA) a prominent band of zinc-containing Timms-stained terminals is present in the inner molecular layer of the ipsilateral dentate gyrus. At 3 months, mossy fibre reorganisation is also seen in the contralateral inner molecular layer of the dentate gyrus and in the infrapyramidal band in the contralateral CA3. The relationship of this reactive plasticity to the CA3 lesion is discussed.

Thinking inside the box. To cope with an increasing disease burden, drug discovery needs biologically relevant and predictive testing systems.

In the next few decades, brain medicine will present a particular socioeconomic challenge for ageing citizens worldwide. Many disorders and ailments that affect the brain—including Alzheimer disease, Parkinson disease, dementia and stroke—are chronic conditions that persist for years or even decades. In addition, many of these disorders have devastating effects, which together create a substantial burden on society: about one‐third of the global disease burden can be attributed to disorders of the brain or nervous system (Olesen & Leonardi, 2003; Andlin‐Sobocki et al , 2005). In the USA alone, there are as many as 5.5 million individuals with Alzheimer disease, 1.5 million with Parkinson disease and 400,000 with multiple sclerosis. These devastating diseases not only place a heavy emotional burden on patients and their care‐givers, but also have an important economic impact. > …about one‐third of the global disease burden can be attributed to disorders of the brain or nervous system Unfortunately, this tremendous socioeconomic problem is worsening, because the risk of suffering from a brain disease increases with age, and life expectancy is increasing worldwide, particularly in developed countries. In the case of Alzheimer disease, for example, ∼10% of US citizens aged over 65 years are affected, the prevalence rate has more than doubled since 1980 and it could increase twofold to threefold by 2050 if the current trend continues (Alzheimers Association, 2006). Unfortunately, research has had little impact on these statistics. Despite vigorous efforts from both the pharmaceutical industry and biomedical researchers, there are still no disease‐modifying treatments available for Alzheimer disease, multiple sclerosis, stroke and a range of other neurological disorders. Some have argued that this problem cannot be solved by standard high‐throughput target‐based drug discovery methods, because we simply do not know which mechanisms to target. Instead, we should revert to previous empirical models of drug …

Stretch-induced injury in organotypic hippocampal slice cultures reproduces in vivo post-traumatic neurodegeneration: role of glutamate receptors and voltage-dependent calcium channels

The relationship between an initial mechanical event causing brain tissue deformation and delayed neurodegeneration in vivo is complex because of the multiplicity of factors involved. We have used a simplified brain surrogate based on rat hippocampal slices grown on deformable silicone membranes to study stretch‐induced traumatic brain injury. Traumatic injury was induced by stretching the culture substrate, and the biological response characterized after 4 days. Morphological abnormalities consistent with traumatic injury in humans were widely observed in injured cultures. Synaptic function was significantly reduced after a severe injury. The N‐methyl‐D‐aspartate (NMDA) receptor antagonist MK‐801 attenuated neuronal damage, prevented loss of microtubule‐associated protein 2 immunoreactivity and attenuated reduction of synaptic function. In contrast, the NMDA receptor antagonists 3‐[(R)‐2‐carboxypiperazin‐4‐yl]‐propyl‐1‐phosphonic acid (CPP) and GYKI53655, were neuroprotective in a moderate but not a severe injury paradigm. Nifedipine, an L‐type voltage‐dependent calcium channel antagonist was protective only after a moderate injury, whereas ω‐conotoxin attenuated damage following severe injury. These results indicate that the mechanism of damage following stretch injury is complex and varies depending on the severity of the insult. In conclusion, the pharmacological, morphological and electrophysiological responses of organotypic hippocampal slice cultures to stretch injury were similar to those observed in vivo. Our model provides an alternative to animal testing for understanding the mechanisms of post‐traumatic delayed cell death and could be used as a high‐content screen to discover neuroprotective compounds before advancing to in vivo models.