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Imaging mummified samples remains a technologic and logistic challenge. CT has been used to successfully image mummified remains noninvasively [1-7]. It was possible to use CT to differentiate cerebral gray and white matter in high-altitude Argentine mummies [4], and Hoffman and Hudgins [5] were able to visualize the desiccated brain in female Egyptian mummies from which the intracranial contents had not been removed. However, MRI, based on water protons, has met with only partial success, with a limited subset of mummified tissues. In early reports of mummy MRI, no signal or image was obtained from a variety of pulse sequences [3, 8]. To obtain MR images from the extremities of a Peruvian mummy, Piepenbrink and coworkers [9] needed to rehydrate the specimen in 20% acetone in water. Although MRI was able to visualize the brain after a corpse had lain 13 months in a river bed, the brain was not fully mummified and showed evidence of putrefaction [10].

An issue that complicates MRI detectability of mummified tissue is the conversion of normal water-laden tissues to adipocere, a firm waxy substance [11]. The formation of adipocere in a desiccated environment (Egyptian desert) compounds the problem compared with moisture-laden peat or bog conditions [12]. Modern chemical analysis within the context of forensic sciences shows that adipocere is the result of hydrolysis of triglycerides into glycerin and free fatty acids [13]. Although this suggests that MRI of an ancient desiccated mummy brain would detect only lipid materials, we found that bound water remained in the sample and that it was possible to generate images of such an ancient brain (Nakht, a 16-year-old weaver from the XX Dynasty) by using fast imaging techniques. Furthermore, multinuclear spectroscopy was able to detect sodium and phosphate ions and numerous organic biochemicals from temporal lobe extracts.

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The first-ever successful MR images of an ancient mummy brain are illustrated in Figure 2 (coronal view) and Figure 3 (axial view). Obtaining these images was technically challenging, and the specimens could be viewed only in the T1-weighted sequence. Despite the waxy appearance of the brain hemispheres consistent with the formation of fatty adipocere, images were formed from residual water in the tissue. Presumably, these water molecules had very short T1 relaxation times because no signal saturation was noted in the T1-weighted images despite very short TR and TE; short T2 relaxation times were also presumed because no signal was detected in the T2-weighted images. The brain images were quite homogeneous in signal intensity and contained surprising internal structure (Figs. 2 and 3). Coronal views (Fig. 2) appeared almost laminar in appearance, perhaps reminiscent of the original cerebral gyri. The internal structure in both imaging planes was complex, with the low-signal areas likely to be air spaces resulting from tissue shrinkage.

The localized proton spectrum of the samples (Fig. 4) revealed two principal water signals in the mummy brain from which the image was obtained appearing at 4.31 and 4.86 ppm. The peak observed at 4.70 ppm in this water-unsuppressed spectrum corresponds to water from the saline phantom (Fig. 4), which was not present for the acquisition of the images shown in Figures 2 and 3. Neither of the resonances at 4.31 and 4.86 ppm nor the smaller peaks observed at 3.64 and 5.23 ppm were indicative of lipid protons.

All three pathologic stains revealed a predominantly homogeneous appearance (Fig. 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H), although numerous hole artifacts were seen in the tissue that could be due to mummification, rehydration, or fixation and processing for histology. In the solochrome cyanin R-stained slides, a uniform composition of the tissue (Fig. 5A) was accompanied by a few hypostained globular areas, some of which displayed a granular appearance (Figs. 5B and 5C). These could be artifactual. In this stain, myelin lipids would have appeared blue, but there was no evidence of such staining. In the H and E-stained sections, eosinophilic structures were observed (Figs. 5D, 5E, 5F). These could be found surrounding some of the holes (Fig. 5D) but were also linear in morphology (Figs. 5E and 5F), reminiscent of vascular structures. Numerous argentophilic granules were observed in the Bielschowsky method-stained slides (Figs. 5G and 5H). These particles were seen in isolation or en masse in large concentrations and could represent calcification resulting from calcium desequestration during mummification. Surprisingly, even after 3,000 years, the mummified and adipocerous brain retained some residual morphologic character on a microscopic level. Because the sample appeared homogeneous on imaging, these microscopic features do not appear to contribute to the MR images.

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Fig. 3 —Axial images of mummy brain hemispheres obtained at 1.5 T show variable signal intensity, irregular pockets of low signal intensity, and complex internal structure.

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Fig. 4 —Localized 2D stimulated echo acquisition mode proton spectrum (1.5 T) of intact mummy brain hemispheres (upper panel) and saline phantom (lower panel) obtained without water suppression. Circular saline phantom can be clearly seen in inset and its signal is at 4.7 ppm in both spectra. Two additional large peaks appear adjacent to water at 4.31 and 4.86 ppm with small shoulders at 3.64 and 5.23 ppm.

The 4.0-T nonlocalized sodium spectrum from the two hemispheres (Fig. 6, upper panel) revealed a single symmetric broad resonance (2,071 Hz, full width at half maximum). No phantom was used for this study and there was an absence of signal when the brain was removed (Fig. 6, lower panel). This result suggests that the sodium ions were in a single-bound environment with a short correlation time (τ_c) in the intact specimen.

NMR spectroscopy on the tissue extractions revealed diverse soluble components. The ³¹P spectrum revealed a single symmetric peak (0.809 ppm) in the D₂O extract (Fig. 7). This observation is consistent with the recovery of phosphate ions from the mummified tissue.

The proton spectrum at 600 MHz in CDCl₃ showed numerous components (Fig. 8). Because we did not treat the samples to remove metal ions, the narrow linewidths observed suggest that the samples did not have paramagnetic contamination. Analysis of the one-dimensional spectrum revealed numerous resonances consistent with the presence of free fatty acids [19]. Peaks at 0.89, 1.24, and 2.31 ppm were consistent with the identification of palmitic and stearic acids and hydroxystearic acid and likely contributed to these resonances and the multiplet observed at 1.60 ppm. Multiplets at 1.99 and 5.35 ppm (double bond) identified residual unhydrogenated oleic acid, and these assignments are annotated on the spectrum (Fig. 8).

There were additional singlets observed at 0.66 and 1.01 ppm, and numerous multiplets of unknown origin at 1.84, 1.95, 4.60, and 7.05 ppm. A singlet was observed at 7.25 ppm due to residual protons in the CDCl₃ solvent. Thus, proton spectroscopy of the CDCl₃ extract revealed the presence of free fatty acids, which are characteristic of adipocere [11], as well as several other biochemicals. When adipocere was found on examination of a corpse after 3-4 months in an aqueous environment, two 9-chloro-10-methoxy(9-methoxy-10-chloro) fatty acids were found to constitute 7.2% of the lipids [20]. We cannot confirm the presence of these fatty acids because two characteristic singlets (3.4 and 3.7 ppm) and two multiplets (3.3 and 4.0 ppm) were absent from the spectrum of our extract.

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Using a strategy that was optimized for tissues with very short water relaxation times, it has been possible to image the internal structures of a mummified Egyptian brain. Because adipocere has been considered to be composed of lipid materials, we assumed that the images would be based on the hydrolyzed lipids. Un-expectedly, the desiccated tissue continued to bear considerable bound water in at least two principal environments, and none of the proton NMR peaks observed from the specimen corresponded to free lipids. Because bound water had a very short T1 relaxation time, it was only possible to visualize the specimens with sequences that were optimized for fast imaging with very short TRs and TEs.

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Fig. 5A —Solochrome cyanin R-, H and E- and Bielschowsky method silver-stained sections of rehydrated right temporal tissue dissected from mummy brain. (All micrographs ×400) Solochrome cyanin R staining. Tissue appears uniform in solochrome cyanin R sections (A) except for occasional lacunar areas showing pale staining (arrows, B and C).

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Fig. 5B —Solochrome cyanin R-, H and E- and Bielschowsky method silver-stained sections of rehydrated right temporal tissue dissected from mummy brain. (All micrographs ×400) Solochrome cyanin R staining. Tissue appears uniform in solochrome cyanin R sections (A) except for occasional lacunar areas showing pale staining (arrows, B and C).

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Fig. 5C —Solochrome cyanin R-, H and E- and Bielschowsky method silver-stained sections of rehydrated right temporal tissue dissected from mummy brain. (All micrographs ×400) Solochrome cyanin R staining. Tissue appears uniform in solochrome cyanin R sections (A) except for occasional lacunar areas showing pale staining (arrows, B and C).

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Fig. 5D —Solochrome cyanin R-, H and E- and Bielschowsky method silver-stained sections of rehydrated right temporal tissue dissected from mummy brain. (All micrographs ×400) Structures in H and E-stained sections (arrows) appear linear in nature and appear to line some of tissue holes. Rest of tissue was uniformly stained.

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Fig. 5E —Solochrome cyanin R-, H and E- and Bielschowsky method silver-stained sections of rehydrated right temporal tissue dissected from mummy brain. (All micrographs ×400) Structures in H and E-stained sections (arrows) appear linear in nature and appear to line some of tissue holes. Rest of tissue was uniformly stained.

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Fig. 5F —Solochrome cyanin R-, H and E- and Bielschowsky method silver-stained sections of rehydrated right temporal tissue dissected from mummy brain. (All micrographs ×400) Structures in H and E-stained sections (arrows) appear linear in nature and appear to line some of tissue holes. Rest of tissue was uniformly stained.

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Fig. 5G —Solochrome cyanin R-, H and E- and Bielschowsky method silver-stained sections of rehydrated right temporal tissue dissected from mummy brain. (All micrographs ×400) Bielschowsky method-stained sections also show dense deposits outlining margins of some holes in tissue (G) with substantial deposits of granular material throughout tissue (H).

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Fig. 5H —Solochrome cyanin R-, H and E- and Bielschowsky method silver-stained sections of rehydrated right temporal tissue dissected from mummy brain. (All micrographs ×400) Bielschowsky method-stained sections also show dense deposits outlining margins of some holes in tissue (G) with substantial deposits of granular material throughout tissue (H).

Paleohistology revealed primarily homogeneous tissue on rehydration. All the samples showed residual tissue shrinkage, which resulted in tissues that had holelike artifacts. Despite this limitation, there were structural anomalies observed in the solochrome cyanin R- and H and E-stained sections. The exact identification of these hypostained lacunar structures and the thin linear eosinophilic threads is unknown. Furthermore, the silver stain revealed a plethora of staining, with some semicircular structures observed that were similar to those stained with eosin. Again, the exact nature of the structures identified is unknown at this time, but calcification is a possible interpretation. Calcium ions would be released from the tissue because the normal proteins, subcellular structures, and membranes were altered during mummification. Similar granular structures have been attributed to Nissl bodies in cresyl violet-stained sections from a desiccated mummified brain [19]. The uniformity of the MR images from the specimen was reflective of the absence of myelin staining in the solochrome cyanin R sections.

Spectroscopy both on the specimens and on extracts revealed additional compositional information. Sodium ions were detected in the specimen and phosphate was extracted using D₂O. In addition, the proton NMR studies of the extract were informative regarding the heterogeneity of available compounds in the biopsied material. Previous studies on the composition of adipocere identify certain specific compounds because the triglycerides are hydrolyzed to glycerol and free fatty acids. At first, stearic and oleic acids, then their metabolites palmitic, hydroxystearic, and oxostearic acids are typically identified in this material [11]. Proton spectroscopy of the extracts was consistent with the detection of these compounds, but gas chromatography coupled to mass spectrometry would be needed to verify the identity of all components of this complex extract. We were unable to confirm the presence of 9-chloro-10-methoxy(9-methoxy-10-chloro) fatty acids [20] in our extract. The characteristics of this sample of adipocere produced in a short time (3-4 months) under water may be different from those produced when the mummy brain was desiccated over 3,000 years. Nonetheless, either insufficient quantities or the solidified structure of the intact specimen prevented detection by proton spectroscopy at 1.5 T using our acquisition parameters.

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Fig. 6 —Upper panel shows 23Na spectrum obtained at 4 T of intact specimens. There is single broad resonance with linewidth of 50 ppm. Lower panel is identical acquisition without brain in coil.

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Fig. 7 —Proton-coupled 31P spectrum obtained at 14 T from deuterium oxide extract of temporal brain shows single resonance at 0.9 ppm with linewidth of 20 Hz. Inset shows spectrum from -3 to 3 ppm.

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Fig. 8 —Proton spectrum at 14 T of deuterated chloroform extract of temporal brain divided into 0.5- to 2.5-ppm and 4.0- to 7.5-ppm sections.

The MRI and MR spectroscopy results obtained at 1.5 T have potential limitations for detection of the fatty component of adipocere. The TR and TE values that were successful in imaging residual bound water may not have been optimal for detection of the lipid components identified in the chloroform extract. Ul-trashort TE imaging [21-23] may have the ability to detect the fatty components of adipocere, particularly if the hydrolyzed fatty acids have a short τ_c. Although the scanner parameters permitted a minimum TE of 1.8 milliseconds at a TR of 9.9 milliseconds, our TE choice of 4.4 milliseconds produced the highest signal for imaging. We were unable to produce any images using T2-weighted sequences that would also need to be further optimized to visualize very short T2 species. Similarly, the in vivo proton spectrum was performed at a TE of 30 milliseconds, although the scanner, which was optimized for patient brain studies, permitted TEs of 15, 30, or 144 milliseconds. It is possible that studies using much shorter TE values could have detected lipid components with shorter T2 or τ_c values [24].

Adipocere is a generic term for the end product of the alteration of the soft tissues of corpses into a grayish-white, soft, creamlike substance, which over time becomes a harder solid and resistant mass. The full decomposition of this material has not been described, but chemical studies are clear that oleic, palmitic, and stearic acids are initially the main constituents, followed by triglyceride hydrolysis, beta oxidation, and hydration of fatty acids to also yield hydroxystearic and oxostearic acids. These constituent changes, accompanied by saponification with calcium and magnesium, can cause solidification of the tissues [11]. This was the type of specimen examined in these studies. The process of mummification under arid and hot conditions in the Egyptian desert produced an unyielding material that was waxlike in consistency. According to Elliot-Smith [25], all of 500 skulls from a prehistorical burial site in Egypt had a preserved brain because of burial in sand in a well-drained location. We observed the same phenomenon with this specimen: mummification by drying with shrinkage [16]. This was also seen in a naturally mummified brain from South Africa [18], in which CT examination was correlated with a total loss of internal structure, as we have seen with MRI. This type of MRI may also be effective in adipocere produced under other ambient conditions because we appeared to image the retained water molecules.

In this study, MRI and multinuclear spectroscopy were used to study cerebral specimens from an ancient mummy to show their morphology and chemical composition and the taphonomic changes that occurred in the desiccated brain. MRI findings were consistent with the uniform appearance of stained sections of specimens extracted from the temporal lobes of the mummy's brain. At a microscopic level, the tissue retained some structures reminiscent of vessel morphology and possible calcium deposits that do not appear visible on the MR images. Although the tissue contained a variety of free fatty acids, they were not detectable in the intact specimen with the acquisition parameters used. We view these studies as works in progress and affirm that MRI and MR spectroscopy can indeed be used to provide additional new insight into mummified tissues.