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A piglet model for detection of hypoxic-ischemic brain injury with magnetic resonance imaging. Early detection kistta hypoxic-ischemic HI injury in the asphyxic newborn is important because present prognostic factors are inadequate. Furthermore, therapeutic interventions may have additional benefit if initiated in time.

Histology and microtubule-associated protein 2 MAP-2 staining was performed in the basal ganglia at the end of the experiment. This piglet model may potentially be used to mimic clinical situations and is suitable for further research investigating HI injury.

Clinical characteristics are not sufficient to determine etiology or prognosis in term infants with encephalopathy, particularly in the absence of a clear history of asphyxia 2. It is well known that diffusion magnetic resonance imaging MRI and proton MR spectroscopy MRS are valid methods for evaluating early alterations in asphyxia, both in human babies and animals 3.

Several studies have shown that MRI is the modality of choice in studies of kisfa injury in term neonates 4 and in newborn piglets 56. Following perinatal hypoxic-ischemic HI injury, proton MRS may be a valuable tool in providing information on timing and pattern of acute brain metabolite changes.

Increased levels of lactate have been detected in neonates with subsequent poor neurodevelopmental outcome and have been characterized as a prognostic factor 7. MR-based diffusion-weighted imaging DWI is sensitive to changes in water diffusion, in both magnitude and direction.

Many studies have shown that the apparent diffusion coefficient ADCas measured by DWI, is reduced in the hyperacute phase following an HI event due to influx of water from the extra- to the intracellular space 8. A previous study has shown that ADC reduction during HI in a piglet model is reversible after 30 min 9. In the setting of acute stroke, fractional anisotropy FA correlates with time of stroke onset Clinical studies conclude that anisotropy measurements are promising for the early detection of impaired brain development in neonates There are only a limited number of studies which have investigated the effect of hypoxia-ischemia on diffusion anisotropy in the acute phase.

In this study, the measurements were performed at baseline and 7 hours after HI. A 7-hour observation period gives us the opportunity to radikulrr changes in the early phases of secondary energy failure, which occurs from 6 to 48 hours after the primary insult 13 The purpose of our study was to describe how MRS and DWI can be added to a piglet model that may potentially be used to mimic clinical situations. Thus, our hypothesis is that these MR techniques are suitable for early assessment of acute tissue changes in the piglet brain after HI damage.

Eadikuler animals had a radikkler SD weight of 3. There were five female and five male piglets. When necessary, a bolus of fentanyl 10 mg or midazolam 1 mg was added. Normoventilation arterial carbon dioxide tension [PaCO 2 ] 4. Inspiratory time of 0. The left femoral artery and vein were cannulated with polyethylene catheters Portex PE, inner diameter 0.

The common carotid arteries on both sides were exposed through a small incision in the neck at the level of the fourth cervical vertebra. The clamping of the common carotid arteries was verified by two-dimensional 2D ultrasonography with color and spectral Doppler. MRI was performed at 1. The anesthetized piglets were imaged with a quadrature extremity coil. MRI was performed prior to hypoxia 15 and 7 hours after the hypoxic event.

The MRI protocol included: Morphological changes on T2- radikuller FLAIR-weighted sequences were evaluated by consensus by two experienced radiologists. The regions of the basal ganglia, cerebral cortex, subcortical white matter, brainstem, and cerebellum were analyzed for low- or high-signal-intensity lesions and absence or presence of edema defined as sulcal and ventricular effacementbefore and after the HI insult Fig.


Morphological changes visualized by T2 in the basal ganglia. The most prominent morphological change seen on the MR images after 7 hours was a slight relative increase in signal intensity in the basal ganglia with effacement of borders post-HI arrows.

A Piglet Model for Detection of Hypoxic-Ischemic Brain Injury with Magnetic Resonance Imaging

From the MRS spectra, four metabolites were measured: Apparent diffusion coefficient ADC maps and fractional anisotropy FA maps were created from the diffusion-weighted images and the diffusion tensor images, respectively, using previously described methods The regions of interest ROI marked with an arrow illustrate the area used for measuring the ADC values in the basal ganglia.

The samples were evaluated by a pathologist. Due to the short time of survival between the hypoxic-ischemic event and the sacrifice, the morphological changes in the basal ganglia were subtle.

Areas with vacuolated neutrophils, shrunken neurons with pyknotic nuclei, and scattered eosinophilic neurons were defined as early necrosis. MAP-2, which is a sensitive marker for neuronal ischemic injury 16was used to confirmthe areas of damage. Necrotic areas showed loss of MAP-2 staining.

For each piglet, a significant difference between values at baseline and after 7 hours was thought to represent an injury. Morphological changes consisting of light global edema were present in four of the 10 piglets on the MRI scans radikulfr 7 hours after the HI insult. Mean ADC values in the raadikuler ganglia for the piglets as a group at baseline and after 7 radkiuler were Apparent diffusion coefficient ADC values in the basal ganglia for the 10 piglets prior to HI gray bars and after 7 hours black bars.

The mean FA values in the basal ganglia were significantly higher after 7 hours compared to baseline 0. Thus, correlation statistics were not calculated for the FA index.

With respect to proton MRS Fig. One of the 10 piglets piglet 6 had no increased lactate after 7 hours, corresponding to the piglet with the lowest relative change in ADC. There was a good correlation between the morphologic changes in the HE-stained sections and loss of MAP-2 staining, as shown in Fig. One piglet piglet 4 showed no histological signs of necrosis. Inset shows eosinophilic neurons arrows40x.

Early necrosis with loss of MAP-2 staining, 10x.

A Piglet Model for Detection of Hypoxic-Ischemic Brain Injury with Magnetic Resonance Imaging

The availability of a well-controlled piglet model for investigating neonatal injury due to HI is of great importance 17and in this study we demonstrate correlation between MR findings and histopatho-logical changes in one such model, with results similar to those of a recent retrospective study of human newborns with HI injury In this study, proton spectra were localized to the basal ganglia because previous studies using MRI have identified this area as vulnerable to hypoxia-ischemia injury in neonates Spectra from this region are also less susceptible to interference arising from extracranial fat tissue Reduced ADC and the presence of lactate were strongly correlated to tissue damage.

Whereas all piglets showed signs of cerebral pathology in the basal ganglia on diffusion-weighted MRI, MRS, or histology after the HI insult, one of the 10 piglets had no increased lactate and another had no histological changes.

Presence of a lactate peak a few hours after birth may be due to residual lactate from the primary insult and may subsequently resolve.

Therefore, the presence of lactate in the first hours after an HI injury may not carry the same adverse prognosis as later elevation seen during secondary energy failure These findings correlate with poor prognosis in clinical studies 22and confirm the important role of proton MRS in assigning an early prognosis of neonates with neonatal encephalopathy It is well known that acute ischemic cerebral changes cannot be reliably identified with these sequences The age of the animals is very important when studying HI.

It has been shown that a difference of just 1 day in rats has a profound impact on the severity of the injury as assessed by spectroscopy and histology We do not know the exact age in hours of each piglet. The piglets were brought to us on the day of the experiment, and they were close to but less than 36 hours old.


Therefore, age variation in the present study may not have affected the results. N iblock et al. The almost instant reduction in tissue water diffusion following cerebral ischemia is well documented and was demonstrated with diffusion-weighted MRI in cats already in DWI has recently been shown to be a very sensitive method to diagnose severe neonatal HI. In a recent study in five neonates with severe HI, ADC was shown to be significantly reduced in the cerebrum relative to the unaffected cerebellum A previous study also observed a gradual increase in ADC toward baseline in the first 2 hours after HI 9.

This may represent transient normalization. In the current study, the persistent reduction in ADC 7 hours after HI is consistent with the histological confirmation of irreversible cell damage. This was also confirmed by the presence of lactate in the basal ganglia at the time of post-HI imaging.

For the piglets as a group, the FA values in the basal ganglia increased 7 hours after HI compared to baseline. As cytotoxic edema develops, there is a shift of water from the extracellular to the intracellular space, but the cell radiuler remains intact and there is no overall increase in tissue water. In our study, three piglets had significantly decreased FA values 7 hours after HI.

The use of MAP-2 immunohistochemistry made it possible to find areas with loss of MAP-2 staining as an indicator of damage, and it was possible to compare the HE-stained areas and the areas with loss of MAP-2 staining to confirm the necrosis seen with light microscopy.

Despite the short time of observation, there were widespread necrotic areas in the basal ganglia. The distribution of brain radijuler has been demonstrated earlier Animal models will always be an approximation to the clinical situation. We used a piglet model because the anatomy and physiology are similar to humans, and the cerebral maturation and myelinization of the newborn pig is comparable to the human neonate A weakness of the current study is the fact that the animals were 12—36 hours old, and therefore to some extent adapted to extrauterine life.

Another limitation is that the current model does not allow long-term follow-up studies in its current form.

Histological findings in this model do, however, confirm considerable brain damage as early as min after the insult V annucci et al. Whether our findings can be applied to detect HI injury in kistta newborn infants should be settled through clinical trials. It is recommended to collect data that enable calculation of brain metabolite concentrations expressed as milli-mole per kilogram wet weight of brain tissue However, only a few studies have measured absolute metabolite concentrations In the current study, it was not technically feasible to perform quantitative spectroscopy.

The combination of neurological assessment and MRS is thought to provide the most reliable prediction of neuron developmental outcome at 1 year of age The fact kusta our experimental model requires general radiluler, and that we do not observe the animals in a wake state, makes neurobehavioral testing impossible.

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Longer observation time and neurobehavioral testing would be desirable for further information. In conclusion, our data correlate with findings in clinical studies, indicating that this piglet model has the potential to mimic clinical situations.

The model is suitable for further research investigating HI injuries in which DWI and MRS can be used to study the effects of early neuroprotective treatment.