W. Bodsch

Recovery of Monkey Brain after Prolonged Ischemia. II. Protein Synthesis and Morphological Alterations

Recovery of protein synthesis following 1 h of complete ischemia of the monkey brain was assessed by 3H-labeled amino acid incorporation in vivo at various postischemic periods between 1.5 and 24 h. The regional autoradiographic patterns obtained were compared on the basis of precursor-product relationships determined biochemically at the end of the tracer incorporation studies. Shortly after ischemia, protein synthesis was severely inhibited, but it gradually recovered with increasing recirculation times. In the cerebellum it returned to almost normal levels within 3 h and in the cortex within 24 h. Hippocampal and thalamic regions, however, did not recover control levels of protein synthesis at 24 h. His-toautoradiographic evaluation of amino acid incorporation in individual neurons revealed recovery of pyramidal neurons in the CA1 and CA3 sectors of the hippocampus within 6 h of recirculation, which, however, was followed by secondary inhibition after longer recirculation. Neurons in cortical layer 5 steadily recovered to near control within 24 h, with the exception of those located in arterial border zones, which returned to only 50% of control at 24 h. Incomplete recovery was also observed in thalamic neurons and Purkinje cells. The regional and histoauto-radiographic pattern of protein synthesis correlated with the morphological appearance of cells. Ischemic cell changes (mainly of the dark type with microvacuolization and perineuronal glial swelling) were marked after short recirculation times but gradually disappeared in parallel with the return of protein synthesis in most regions of the brain. Only in pyramidal cells of the hippocampus, thalamic neurons, and Purkinje cells were changes not reversed during the observation period. The results obtained corroborate the electrophysiological observations reported in the first part of this investigation and support the notion that the majority of the neurons of monkey brain survive complete cerebrocirculatory arrest of 1 h for at least 1 day.

A quantitative regional analysis of amino acids involved in rat brain protein synthesis by high performance liquid chromatography.

Abstract: A biochemical method is described for the simultaneous quantitative estimation of unidirectional blood‐brain amino acid influx and protein biosynthesis in individual structures of the rat brain. The method involved a double labeling experiment started by the administration of [14C]carboxyl‐labeled amino acids and terminated 2 min after infusion of 3H‐labeled amino acids, each at tracer quantities, the total labeling period being 45 min. Specific radioactivities of 14C‐ or 3H‐labeled phenyl‐alanine, tyrosine, leucine, isoleucine and valine were determined in plasma and in small brain tissue samples for free amino acids, aminoacyl‐tRNAs and proteins. Amino acids were converted to their corresponding 5‐dimethylamino‐naphthalenesulfonyl (Dns, dansyl) derivatives and separated on HPLC C18 reversed‐phase columns isocratically according to a newly developed optimizing procedure. The order of influx values between the neutral amino acids in relation to each other was Leu > Tyr > Ile > Phe > Val in every structure examined. Although aminoacylation of tRNAs was found to proceed to a comparable degree for neutral amino acids in all regions investigated, the specific radioactivity of amino acids attached to tRNAs differed substantially from that in the free amino acid pool, especially for leucine and valine. The results indicate the necessity of aminoacyl‐tRNA determinations for tracer incorporation studies in protein synthesis analysis. Relative protein synthesis rates in the halothane‐anesthetized rat were determined to be 30 and 67–91 pmol total amino acid incorporation/min/mg tissue for white and gray matter, respectively.

Triple-tracer autoradiography of cerebral blood flow, glucose utilization, and protein synthesis in rat brain.

A triple-tracer autoradiographic technique is described that permits the simultaneous measurement of cerebral blood flow, glucose consumption, and protein synthesis using 131I-iodoantipyrine (131I-IAP), [14C]deoxyclusively from 14C disintegrations without contamination by either 131I or 3H and that represented regional glucose utilization. Brain sections were then wash-incubated for 12 h to remove [14C]DG, [14C]DG-6-phosphate, and free 3H-amino acids from the tissue, and exposed to 3H-sensitive LKB Ultro-film for 2 weeks for autoradiography of 3H-amino acid incorporation into proteins. 14C radioactivity remaining in the tissue section after wash-incubation was determined by exposing sections again for 2 weeks to Kodak NMB film; the resulting contribution to the blackening of 3H-autoradiograms was corrected for by means of digital subtraction using an image-processing system. The triple-tracer autoradiographic technique was validated in rats under various physiological and pathophysiological conditions. In intact animals extinction correction was necessary only for 3H-autoradiograms. Under pathophysiological conditions, however, significant contamination of 131I by 14C occurred in regions with low blood flow and increased glucose utilization rate; this also required correction by digital subtraction. The interpretation of triple-tracer autoradiographic results is limited by the same restrictions as single-tracer autoradiography, but the simultaneous assessment of the three parameters considerably facilitates the interpretation of the flow/metabolic relationship, particularly under pathological conditions.

Immunochemical method for quantitative evaluation of vasogenic brain edema following cold injury of rat brain

An immunochemical method is described for quantitative assessment of serum proteins and hemoglobin content in brain tissue homogenates. Using a combination of affinity chromatography and radioimmunoassay, the sensitivity of the method is 50 ng hemoglobin and 100 ng serum protein per assay, respectively. The method was used to measure cerebral hematocrit, blood volume and serum protein extravasation in rat brain at various times following cold injury. In control rats cerebral blood volume was 6.88 +/- 0.15 ml/100 g and cerebral hematocrit 26.4 +/- 0.86% (means +/- S.E.). Following cold injury blood volume did not significantly change, but there was a gradual increase of extravasated serum proteins, reaching a maximum of 21.54 +/- 2.76 mg/g d.w. after 8 hours. Thereafter protein content gradually declined, but even after 64 h it was distinctly increased. Protein extravasation was partly dissociated from the increase of brain water and sodium which reached a maximum already after 2 h and which normalized within 32 and 64 h, respectively. It is concluded that edema fluid associated with cold injury is not simply an ultrafiltrate of blood serum but consists of cytotoxic and vasogenic components which follow a different time course both during formation and resolution of edema.

Biochemical and Autoradiographic Study of Cerebral Protein Synthesis with [18F]- and [14C]Fluorophenylalanine

Fluorine‐18‐labeled ortho or para isomers of l‐fluorophenylalanine were used in double‐label experiments together with l‐[3H]phenylalanine for amino acid incorporation into cerebral proteins of Mongolian gerbil brain. It was demonstrated by qualitative regional comparison of the 18F and 3H autoradiographic images that l‐p‐[18F]fluo‐rophenylalanine is incorporated into proteins and exhibits a regional cerebral protein synthesis pattern. To a minor extent, l‐p‐fluorophenyl[3–14C]alanine and l‐o‐[18F]fluo‐rophenylalanine are hydroxylated in vivo to form labeled tyrosine or tyrosine analogues that are incorporated into cerebral proteins as well. The advantage and validity of the application of l‐p‐[18F]fluorophenylalanine with positron emission tomography for noninvasive studies of cerebral protein synthesis in humans are evaluated on the basis of an experimental animal approach.

Regional cerebral blood flow, glucose metabolism, protein synthesis, serum protein extravasation, and content of biochemical substrates in stroke-prone spontaneously hypertensive rats.

Stroke-prone spontaneously hypertensive rats with arterial blood pressure above 210 mmHg were taken for the present study after appearance of neurological symptoms. Regional cerebral blood flow, glucose metabolism, and protein synthesis rate were evaluated on the same brain section by means of triple-labelled autoradiographic techniques. Consecutive sections were used in the pictorial presentation of glucose, ATP, and serum protein extravasation. In addition, NADH-fluorescence was recorded. Two different patterns of hypertension-induced brain lesions could be distinguished: in two animals sharply demarcated cysts were visible in the cortical grey matter. In these animals no regional inhomogeneities of flow and metabolism were present remote from the infarct. In contrast, in three animals cysts were located in the white matter, leading to pronounced hemodynamic and metabolic disturbances throughout the brain. It is concluded that edema-induced brain swelling was the main cause for reduction in blood flow and metabolism.

125I-antibody autoradiography and peptide fragments of albumin in cerebral edema.

Abstract: The regional distribution of albumin in serum extravasations of cerebral edema was visualized on intact brain slices by autoradiography of 125I‐labeled antibodies directed against albumin. Following autoradiographic imaging of edema protein spread, concentrations of total serum proteins were determined by radioimmunoassay in tissue micro samples taken from various regions of the brain. Peptide fragments of albumin—produced upon proteolytic breakdown of the native protein in vivo— were separated by affinity chromatography and HPLC. The combination of techniques for imaging, direct quantification, and analysis of molecular structure of serum proteins was proved to be valid in three different types of experimental cerebral edema in the rat: cortical cryogenic lesion, brain tumors, and stroke‐prone spontaneous hypertension. The results indicate differences in the reactivity of edematous tissue with respect to proteolytic activity, depending on the susceptibility of serum proteins to in vivo fragmentation.

Pathophysiological Aspects of Blood-Brain Barrier Disturbances in Experimental Brain Tumors and Brain Abscesses

Experimental tumors and abscesses were produced by intrahemispheric inoculation of a blastomatous glial cell clone and of staphylococcus aureus, respectively. In both models severe vasogenic brain edema developed. The site of the barrier lesion was identified by systemic application of Evans blue or peroxidase, and the spread of edema by immunoautoradiographic localisation of extravasated serum proteins. In both experimental conditions, serum proteins accumulated diffusely in the white matter of the ipsilateral hemisphere, although the barrier lesion was strictly confined to the pathological focus. Water content of the edematous white matter in the vicinity of tumors and abscesses increased from 69.1 to 80.6 and 82.3 ml/100g w.w., respectively. This increase was associated with a volume-dependent decrease of flow, a parallel increase of sodium and an increase of extravasated serum proteins. The latter was determined by a newly developed immunochemical approach with appropriate corrections for the intravascular fraction of total serum protein content. The calculated concentration of sodium in edema fluid of tumors and abscesses amounted to 132 and 129 ueq/ml, respectively. The concentration of serum proteins was 8.7 and 6.4 mg/ml, respectively. Protein content of edema fluid, in consequence was less than 10% of blood serum. This suggests that fluid accumulation in vasogenic edema cannot be explained by the oncotic properties of extravasated proteins alone.

Local Rates of Cerebral Protein Synthesis in the Gerbil and Monkey Brain

In autoradiographic studies of cerebral metabolism a radioactively labeled precursor is commonly used to trace the events of neurochemical reactions following brain uptake of the isotopic compound. Therefore, knowledge of the biochemical mechanisms operative within the observation period, i.e., the labeling period, is essential for any attempt to quantify reaction rates. These may be predicted to a certain extent from the integration of the specific radioactivity input function to the system and from the amount of radioactivity in brain per unit mass of tissue. The validity of this approach is largely dependent on the complexity of the mechanism under investigation and may, therefore, require more or less extensive additional measurements or assumptions.

Regional Quantitative Biochemistry and Autoradiography of Protein Synthesis and Serum Extravasation in Brain Edema

Since cerebral metabolic processes are mediated and controlled by enzymes and regulating peptides such as neurohormones, there is considerable interest to relate altered synthesis rates of single specific proteins to the functional activity in various cerebral structures, especially in altered physiological states. The basic mechanisms underlying changes in protein synthesis can be analyzed on a regional level by investigation of the total cellular protein biosynthesis in vivo by radioactive amino acid incorporation2. Coronal sections subjected to autoradiography for regional visualization of in vivo labeled proteins may be further utilized for demonstration of single specific proteins by direct localization of their immunoreactive sites with radioactively labeled antibodies. A correlation can also be carried out with regional blood flow and the energy state of the brain, using appropriate autoradiograph-ic10,9, bioluminescent6,8 and fluoroscopic11 techniques.